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There were different tumor cell clones between LN1 and LN2.  A,  CT and the...
Published: 28 July 2022
FIGURE 1 There were different tumor cell clones between LN1 and LN2. A, CT and the pathology of a male in his late 60s. Before the initiation of treatment, LN1 and LN2 were located next to each other (arrows), which were subsequently resected surgically. Computed tomography (top) and HE staining (bottom) are presented. Scale bars, 2 mm. B, IHC. FFPE sections (3 μm) from LN1 and LN2 were stained. Representative CD8 and PD-1 double staining and the quantified summary are presented. Scale bars, 50 μm. C, Representative driver gene alterations. Extracted DNA from LN1 and LN2 were sequenced and representative driver gene alterations are presented. CN, copy number. D, Clonal evolution of tumor cells. The cellular prevalence of clones carrying individual nonsynonymous mutations in LN1 and LN2 was determined using PyClone. The determined cellular prevalence was used as input, and the phylogenetic relationships of clones were inferred with LICHeE. The means and SDs are shown. A t test was used to calculate statistical significance in B . **, P < 0.01. More
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LN1, but not LN2, had skewed exhausted T-cell clones in the TME.  A,  T-cel...
Published: 28 July 2022
FIGURE 2 LN1, but not LN2, had skewed exhausted T-cell clones in the TME. A, T-cell clustering. We digested LN1 and LN2 to extract TILs. Single-cell sequencing was performed for sorted CD3+ T cells from the extracted TILs. The data of two TIL samples were merged and then clustered based on gene expression. UMAP figure (left) and the summary (right) are presented. B, Representative gene expression in each cluster. Representative genes that are generally used for annotation are shown. C, T-cell clonotype diversity. Pielou's evenness indexes of each sample and each cluster are calculated and summarized. Bars indicate the indexes of all clusters. D, Top 10 clonotype distributions from each sample. The distribution of the top 10 clonotypes from each TIL sample in the UMAP figure is presented. More
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An overlapped exhausted T cell clonotype responded to an overlapped neoanti...
Published: 28 July 2022
FIGURE 3 An overlapped exhausted T cell clonotype responded to an overlapped neoantigen. A, Venn diagrams of nonsynonymous somatic mutations (TMB) and predicted neoantigens. WES for LN1 and LN2 was performed. We used the NetMHCpan algorithm version 4.0 in order to predict the neoantigen candidates from the WES data ( 38, 39 ). The numbers of nonsynonymous somatic mutations (left) and predicted neoantigens with strong binding (right) are shown. B, Clonotype in LN1 and LN2. We merged the clonotypes of LN1 with LN2, which were grouped into LN1 dominant, equivalent, or LN2 dominant clonotypes according to their frequencies. The UMAP figures of clonotype groups (left) and exhausted T cell frequency (right) are shown. Arrows, tested clonotypes in Supplementary Table S7 . C, Peptide assay. After mutated IMPA2 peptide pulse, TCR-transduced NFAT-Luc-Jurkat cell lines were cocultured with autologous cells for 24 hours. Thereafter, luciferase activity was analyzed. The fold change in each NFAT-Luc-Jurkat cell line with no peptide pulse (DMSO) is presented. All in vitro experiments were performed in triplicate, and we show the means and SEMs. To calculate statistical significance in C, one-way ANOVA with the Bonferroni correction were used. *, P < 0.05; ns, not significant. More
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Mouse tumors from different clones had different TME.  A,  Graphic of the e...
Published: 28 July 2022
FIGURE 4 Mouse tumors from different clones had different TME. A, Graphic of the experimental schema of in vivo experiments. We created several single tumor cell clones from the same mouse tumor cell line and injected them subcutaneously. B, IHC for CD8. FFPE sections (3 μm) of tumor samples from each clone were stained. Representative CD8 staining is presented. C and D, PD-1 expression by CD8+ T cells ( C ) and IFNɤ+PD-1+CD8+ T cells ( D ) in the TME. Representative flow cytometry staining (left) and the summaries (right) are shown. All in vivo experiments were performed in duplicate and produced similar results. The means and SEMs are shown. T-tests were used to calculate statistical significance in B , C , and D . **, P < 0.01; ***, P < 0.001. More
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Mouse tumors from different clones exhibited mixed responses to PD-1 blocka...
Published: 28 July 2022
FIGURE 5 Mouse tumors from different clones exhibited mixed responses to PD-1 blockade. A, Venn diagrams of nonsynonymous somatic mutations (TMB) and predicted neoantigens. WES for MC-38 (parent), MC-38 #1C11, and MC-38#2D10 was performed. We used the NetMHCpan algorithm version 4.0 in order to predict the neoantigen candidates from the WES data ( 38, 39 ). The numbers of nonsynonymous somatic mutations (left) and predicted neoantigens with strong binding (right) are shown. B, Clonal evolution of tumor cells. The cellular prevalence of clones with individual nonsynonymous mutations in parental MC-38, #1C11, and #2D10 was determined with PyClone. As input, the determined cellular prevalence was used, and the clonal phylogenetic relationships were inferred using LICHeE. C, TCR clustering. Bulk RNA were extracted from tumors and sequenced for TCR and the results were clustered. D, Efficacy of PD-1 blockade in a mouse model. Cells (1 × 106) were subcutaneously inoculated in immunocompetent wild-type mice, and tumor volume was measured twice a week. We grouped mice when the tumor volume reached approximately 100 mm3 (n = 6 per group). Afterward, anti-PD-1 mAb or control mAb was administered intraperitoneally three times every 3 days. All in vivo experiments were performed in duplicate with similar results. We show the means and SEMs. To calculate statistical significance in D, two-way ANOVA with Bonferroni correction were used. **, P < 0.01; ns, not significant. More
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Mixed responders to immunotherapies had a poor prognosis compared with nonm...
Published: 28 July 2022
FIGURE 6 Mixed responders to immunotherapies had a poor prognosis compared with nonmixed responders in various cancer types. A, The frequencies of mixed responses in various cancer types. We investigated the clinical data in several cancer types, including melanoma, NSCLC, gastric cancer, and head and neck cancer, treated with PD-1 blockade monotherapies. A mixed response was defined as previously reported ( 33 ). The frequencies according to cancer types (left) or RECIST response (right) are presented. B and C, Survival curves. We compared a prognosis in all patients among nonmixed responders, nonmixed nonresponders, and mixed responders. A prognosis in mixed responders was also compared according to local therapies. PFS and OS were defined as the time from the initiation of PD-1 blockade therapies until the first observation of disease progression or death from any cause and the time from the initiation of PD-1 blockade therapies until death from any cause, respectively. PFS (left) and OS (right) in all patients according to responses ( B ) and OS in mixed responders according to local therapies ( C ) are presented. PFS and OS were analyzed using the Kaplan–Meier method and compared among groups using a log-rank test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. More
Journal Articles
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Loss of vascular endothelial GLS reduces breast cancer growth.  A,  A schem...
Published: 21 July 2022
FIGURE 1 Loss of vascular endothelial GLS reduces breast cancer growth. A, A schematic for inducible EC-specific GLS knockout model (GLSECKO). B, Western analysis and qRT-PCR of mouse lung microvascular ECs showing GLS loss in GLSECKO samples. C, Dual immunofluorescence images and quantification showing GLS (green) in CD31+ (red) blood vessels of E0771 tumors grown in WT versus GLSECKO mice (n = 4 mice per group; Scale bar: 200 μm). Light blue arrows, GLS+ tumor ECs. D, Intracellular glutamate concentration measured from lung microvascular ECs isolated from GLS f/f mice and treated with Ad-control (WT) or Ad-Cre (GLSECKO). E, E0771 tumor growth curves. P = 0.0225, two-way ANOVA with Sidak multiple comparisons correction test. F, End-stage E0771 tumor weight in WT and GLSECKO mice (n = 15–18). G, Cleaved-caspase quantification in WT versus GLSECKO E0771 tumors (n = 10–13 mice per group). H, Ki-67 quantification in WT versus GLSECKO E0771 tumors (n = 6–7 mice per group). I, MMTV-PyMT tumor growth curves (n = 11–12), P = 0.0001, two-way ANOVA with Sidak multiple comparisons correction test. J, End-stage MMTV-PyMT tumor weight in WT and GLSECKO mice (n = 11–12). All data are presented as mean ± SEM from two or three independent experiments. P values of B , C , D , F , G , H , and J were determined by two-tailed unpaired Student t test. More
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Loss of vascular endothelial GLS reduces breast cancer metastasis.  A,  A s...
Published: 21 July 2022
FIGURE 2 Loss of vascular endothelial GLS reduces breast cancer metastasis. A, A schematic diagram showing the experimental procedure of tamoxifen treatment (5×: five times) and E0771 mammary fat pad implantation. B, Representative histologic images of H&E-stained lung sections from WT and GLSECKO mice (n = 9–10). Scale bar: 200 μm. Blue arrows denote lesions. C, Quantification of metastatic lung lesions per mouse in WT versus GLSECKO (n = 9–10 mice per group). D, A schematic diagram showing the experimental procedure of tamoxifen treatment (five times), E0771 mammary fat pad implantation, resection, and India ink Injection. E, Representative images of India ink–infused lungs from WT versus GLSECKO mice following primary tumor resection. Red arrows indicate metastatic foci. H (blue) indicates the position of heart. F, Quantification of metastatic nodules in lungs of WT versus GLSECKO (n = 9 mice per group). Data in C and F are presented as mean ± SEM from two or three independent experiments. P values were determined by two-tailed unpaired Student t test. More
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Endothelial GLS deletion reduces tumor vascular density and normalizes tumo...
Published: 21 July 2022
FIGURE 3 Endothelial GLS deletion reduces tumor vascular density and normalizes tumor vessels. A, Representative immunofluorescent images and quantification of CD31+ vessels (red), DAPI (nuclei, blue) in E0771 tumors harvested from WT control and GLSECKO mice (n = 10–12 mice per group). Scale bar: 100 μm. B, Representative immunofluorescent images and quantification of pericyte coverage (α-SMA; green) on tumor vessels (red). Pericyte coverage is presented as percentage of α-SMA+/CD31+ blood vessels (yellow), (n = 7–8 mice per group). Scale bar: 100 μm. C, Representative immunofluorescent images and quantification of pericyte coverage on tumor vessels using NG2 marker (green). n = 7 per group. Scale bar: 50 μm. D, Representative immunofluorescent images and quantification of tumor vessel perfusion. Functional blood vessels were assessed by perfusion of FITC-lectin (green) in CD31+ tumor blood vessels (red). Vessel perfusion is presented as percentage of Lectin+ area within the CD31+ vessels (n = 5 per group). Scale bar: 100 μm. E, Representative immunofluorescent images and quantification of pimonidazole+ hypoxic regions within E0771 tumors. Hypoxia area was assessed by injecting hydroxyprobe into tumor-bearing mice. Hypoxic regions (green) and CD31+ blood vessels (red) are shown within the tumor (n = 8 mice per group). Scale bar: 100 μm. All data are presented as mean ± SEM from two or three independent experiments. P values were determined by two-tailed unpaired Student t test. More
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Leptin treatment rescues tumor growth defects in GLS<sup>ECKO</sup> mice.  ...
Published: 21 July 2022
FIGURE 4 Leptin treatment rescues tumor growth defects in GLSECKO mice. A, Cytokine array showing differentially expressed leptin and leptin receptor in WT versus GLSECKO E0771 tumor lysates. B, Leptin and Leptin receptor mRNA expression was measured by qRT-PCR (n = 3–4) in E0771 tumor cells isolated from WT versus GLSECKO mice. C, Leptin ELISA from whole tumor lysates of WT versus GLSECKO (n = 5 mice per group). D, A schematic diagram of experimental design with E0771 tumor allograft and leptin treatment (1 mg/kg). E, Tumor volume change of WT and GLSECKO mice treated with leptin or PBS control (n = 8–13) calculated as [(VfinalVinitial)/Vinitial]. Vfinal = volume on last day of treatment; Vinitial = volume on first day of treatment. F, Quantification of tumor volume change. Representative images ( G ) and quantification of α-SMA+CD31+ vessels ( H ), showing tumor vessel normalization reversal following leptin treatment. (Scale bar: 50 μm). All data are presented as ± SEM. P values were determined by two-tailed unpaired Student t test ( B and C ), one-way ANOVA with Tukey multiple comparisons test ( F and H ). More
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Loss of endothelial GLS enhances the efficacy of chemotherapeutic agents.  ...
Published: 21 July 2022
FIGURE 5 Loss of endothelial GLS enhances the efficacy of chemotherapeutic agents. A, A schematic diagram showing the experimental procedure of tamoxifen (Tam) treatment (five times), tumor implantation, and DOXO treatment. B, Representative images and quantification of DOXO autofluorescence in E0771 tumors grown in WT versus GLSECKO mice (n = 13–14 mice per group). The doxorubicin+ area (DOXO green) is presented as a percentage of total nuclei, DAPI+ (blue). Scale bar: 200 μm. C, A schematic diagram showing the experimental procedure of tamoxifen treatment (five times), tumor inoculation, and Cpt treatment. D, Growth curve of E0771 tumors grown in WT versus GLSECKO mice treated with either vehicle control (PBS) or Cpt, n = 6–7 mice per group. **, P ≤ 0.01. *, P ≤ 0.05. E, Representative histologic images of H&E-stained lung sections of Cpt and control treated WT and GLSECKO tumors (n = 6–7 mice per group). Blue arrows indicate metastatic foci. Scale bar: 200 μm. Quantification of metastatic lung lesions per mouse in WT versus GLSECKO treatment groups is shown in Supplementary Fig. S5 . F, A schematic diagram showing the experimental procedure of tamoxifen treatment, tumor inoculation, CB-839, and Cpt treatment. G, Growth curve of E0771 tumors upon treatment with vehicle (Veh), CB-839 (50 mg/kg by i.p, daily), and Cpt (4 mg/kg by i.p, every other day), alone or in combination with CB839 (n = 6–7 per group). All data are presented as mean ± SEM from two or three independent experiments. P values were determined by two-tailed unpaired Student t test ( B ), two-way ANOVA with Tukey multiple comparison test ( D and G ). **, P ≤ 0.01. *, P ≤ 0.05. More
Journal Articles
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Design and analysis of mUNO-targeted St-PGA.  A,  Representative structure ...
Published: 28 June 2022
FIGURE 1 Design and analysis of mUNO-targeted St-PGA. A, Representative structure of St-PGA decorated with mUNO peptides (red) and OG (green). B, DLS graph demonstrating uniform size for both St-PGA-OG-mUNO and St-PGA-OG. C, A snapshot of modeled St-PGA structure in water and Na+ counterions at the last stage of the simulation (50 ns), displaying the three arms in different colors for visual clarity. The average gyration radius was 5.6 ± 0.3 nm, t shows time in ns. D, Representative MD snapshot of a single St-PGA-mUNO branch containing three equidistant mUNO peptides. Green spheres represent mUNO and a Licorice representation shows the linker. E, mUNO rotation around the PGA chain for each of the three peptides (black, red, and green lines). F, PGA chain secondary structure evolution, where red and brown regions show how mUNO perturbs the chain structure, turning alpha helices into random coils. More
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St-PGA-OG-mUNO targets CD206<sup>+</sup> TAMs in models of orthotopic TNBC ...
Published: 28 June 2022
FIGURE 2 St-PGA-OG-mUNO targets CD206+ TAMs in models of orthotopic TNBC and experimental metastasis of TNBC and displays an extended plasma half-life. Homing studies with intraperitonally administered St-PGA-OG-mUNO (0.41 mg/0.5 mL of PBS) or St-PGA-OG (0.35 mg/0.5 mL of PBS), after 6 hours of circulation. N = 3 for orthotopic TNBC and N = 2 for experimental metastasis of TNBC. A–G, Homing in orthotopic TNBC. A, St-PGA-OG-mUNO displayed high colocalization between OG and CD206 (yellow signal), whereas St-PGA-OG displayed minimal colocalization ( B ). C, Graph depicting the quantification of CD206 and OG colocalization in the orthotopic TNBC. St-PGA-OG-mUNO and St-PGA-OG did not show any homing to CD86+ cells (M1 macrophages; D and E ) nor CD11c+ cells (DCs; F and G ). H–N , Homing study in the experimental metastasis of TNBC. St-PGA-OG-mUNO displayed high colocalization with OG and CD206 (yellow signal; H ), whereas St-PGA-OG showed minimal colocalization ( I ). J, Graph depicting the quantification of CD206 and OG colocalization in the experimental metastasis of TNBC. St-PGA-OG-mUNO and St-PGA-OG did not show any homing to CD86+ cells (M1 macrophages; K and L ) or CD11c+ cells (DCs; M and N ). Scale bars = 20 μm. O, Quantification of colocalization analysis for St-PGA-OG-mUNO or FAM-mUNO with CD206 homing after 6 hours of circulation, N = 2 (30 nanomoles in OG and FAM, respectively). Colocalization was quantified using the Fiji program and Pearson coefficient (for more information, see Materials and Methods). P, Mean OG/FAM signal per CD206+ cell analyzed using the ImageJ program. Q, Plasma fluorescence (in the green channel) of intraperitoneallly administered St-PGA-OG-mUNO (dose 15 nanomoles in OG) in healthy Balb/c mice (N = 3). R, Rat anti-mouse PD-L1 was intravenously injected 10 days after tumor induction (p.i.) and circulated for 24 hours after which time, mice were sacrificed, tumors collected, fixed, and stained for rat IgG. S, PDL1 expression was detected in noninjected subcutaneous 4T1 tumors by staining with rat anti-mouse PDL1. T and U, Representative images showing St-PGA-OG-mUNO (0.41 mg/0.5 mL) intraperitoneally injected 10 days after tumor induction and circulated for 6 hours after which time, mice were sacrificed, tumors fixed, and then stained for OG and CD206. The OG channel is shown separately in T , and the colocalization with CD206 for the same image is shown in U . Scale bars = 50 μm. Error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01. More
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OximUNO enhances the <em>in vitro</em> efficacy of DOX on M2-resemb...
Published: 28 June 2022
FIGURE 3 OximUNO enhances the in vitro efficacy of DOX on M2-resembling macrophages. A, Simplified form of OximUNO (left) and molecular structure (right) showing St-PGA decorated with mUNO (red) and DOX (green). B, A DLS graph for measurements shown in Supplementary Table S2 , indicating the uniform size of OximUNO and St-PGA-DOX. C, DOX release from OximUNO showing the drug release in PBS, intraperitoneal fluid, acetate buffer or in the presence of cathepsin B. D, CD206 expression by M1-resembling (green) and M2-resembling (red) macrophages using FC. In vitro cytotoxicity in primary human M2-resembling (MCSF + IL4 polarized; E ), M1-resembling (MCSF + IFNγ + LPS polarized; F ) macrophages after treatment with OximUNO (red bars), St-PGA-DOX (blue bars), and DOX (purple bars) following a 15-minute incubation, washed, cultured for additional 48 hours, and then analyzed for cell viability as evaluated by MTT assay. Error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. More
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OximUNO treatment reduces primary tumor growth and pulmonary metastases and...
Published: 28 June 2022
FIGURE 4 OximUNO treatment reduces primary tumor growth and pulmonary metastases and alleviates immunosuppression. Treatment with OximUNO, St-PGA-DOX, or DOX at 2 mg/kg of DOX in mice bearing orthotopic TNBC tumors (N = 5). Intraperitoneal injections began when tumors reached 25 mm3 and were performed every other day to give a total of nine injections. A, Primary tumor volume progression during treatment. Orange arrows indicate injection days. B, Primary tumor weight at the experimental endpoint, demonstrating a significantly smaller weight for OximUNO-treated mice (red bar) than other groups. C, Mouse bodyweight analysis suggests the safety of OximUNO treatment (red line); meanwhile, DOX treatment induced a significant reduction in bodyweight by the experimental endpoint (purple line). Dark gray ∗ DOX versus PBS, orange ∗ DOX versus St-PGA-DOX. D, Representative H&E images showing spontaneous pulmonary metastases for all groups (scale bars = 400 μm); OximUNO treatment associated with the smallest metastatic area ( E ) and the lowest number of average nodules per lung ( F ). G, Representative images showing the expression of CD31 and blood vessels in primary tumors. H, Graph depicting the expression of CD31 in primary tumors. I, Graph depicting the CD31+ blood vessel count in primary tumors. CD31 expression and blood vessel count were calculated using ImageJ and five images per mouse per group for expression analysis and at least three images per mouse per group for blood vessel count. Representative confocal microscopy images and quantification graphs of primary tumors demonstrating the expression of CD206 ( J and K) , CD8 ( L and M ), and FOXP3 ( N and O ). Scale bars = 50 μm. P, Graph of CD8/FOXP3 expression ratio showing a shift in the immune profile. Quantification was performed using the ImageJ from at least three images per mouse and 5 mice per group. Error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., > 0.05. More
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OximUNO treatment in experimental metastasis of TNBC significantly reduces ...
Published: 28 June 2022
FIGURE 5 OximUNO treatment in experimental metastasis of TNBC significantly reduces CD206+ TAM number and tumor burden and alleviates immunosuppression. Treatment with OximUNO, St-PGA-DOX, or DOX at 2 mg/kg of DOX in the experimental metastasis of TNBC model, created using GFP-labeled 4T1 cells (N = 6). Intraperitoneal injections began on day 4 p.i. and were performed every other day to give a total of six injections. A, Quantification of whole lung GFP fluorescence at the experimental endpoint using the ImageJ (N = 6). B, Representative macroscopic photographs of GFP fluorescence in the lungs. C, Representative confocal microscopy images of GFP expression, scale bars = 100 μm. D, Representative H&E images showing pulmonary metastases for all groups (scale bars = 900 μm). E, Quantification of pulmonary metastases from H&E images, expressed as percentual area of whole lung section. F, Mouse bodyweight analysis, demonstrating significantly lower bodyweight lost with OximUNO (red line) compared with St-PGA-DOX–treated mice (blue line) and DOX-treated mice (purple dotted line). Orange arrows indicate injection days. G–J , FC analysis on three right lungs per group. M2 TAMs (CD206+; G ), M1 TAMs ( H ), CTLs ( I ) and Tregs ( J ). Representative IF images and analysis on the pulmonary tumor nodules to detect the expression of CD206 ( K and L ), CD8 ( M and N ), and FOXP3 ( O and P ). Q, Graph showing CD8/FOXP3 expression ratio. IF images quantified using the ImageJ from at least five images per mouse and 3 mice per group. Scale bars = 50 μm. Error bars represent SEM. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; n.s., > 0.05. More
Journal Articles