Journal: Advanced Science

Article Title: Mapping the Tissue‐of‐Origins of Mesenchymal Stromal Cells in Injury Repair

doi: 10.1002/advs.202509533

Figure Lengend Snippet: BM‐MSC‐derived myofibroblasts or CAFs were not detected in distal tissue fibrosis or tumors. A) Confocal imaging of kidney sections from normal and UUO‐induced renal fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. B) Confocal imaging of lung sections from normal and bleomycin‐induced pulmonary fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. C) Confocal imaging of liver sections from normal and CCl 4 ‐induced liver fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. D) Confocal imaging of liver sections from normal and DDC‐induced liver fibrotic Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Myofibroblasts were indicated with anti‐Col1 antibody staining. E–H) Quantification of the percentages of DAPI + Col1 + myofibroblasts that were ZsGreen + and tdTomato + in renal fibrosis (E), pulmonary fibrosis (F), CCl 4 ‐induced liver fibrosis (G) and DDC‐induced liver fibrosis (H). n = 5 mice from 4 independent experiments. I) Confocal imaging of E0771‐induced subcutaneous tumors from Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. Cancer associated fibroblasts (CAFs) were indicated with anti‐Col1 antibody staining. J) Confocal imaging of normal colons and AOM/DSS‐induced colorectal cancer from Pdgfra creER ;Sp7 dre ;R26 ZT1 mice treated with tamoxifen at 2 months old. CAFs were indicated with anti‐Col1 antibody staining. K,L) Quantification of the percentages of DAPI + Col1 + CAFs that were ZsGreen + and tdTomato + in subcutaneous tumors (K) and colorectal cancer (L). n = 3 mice from 3 independent experiments.

Article Snippet: The E0771 mouse breast cancer cell line (CRL‐3461, RRID:CVCL_GR23) was obtained from the American Tissue Type Collection (ATCC) in 2018.

Techniques: Derivative Assay, Imaging, Staining

Journal: bioRxiv

Article Title: Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

doi: 10.1101/2025.11.23.690059

Figure Lengend Snippet: (A) Primary BM-DC/MΦs were differentiated from WT and Arc⁻/⁻ mice. WT cells were either transfected with Arc and A5U-GFP RNAs (Arc⁺ group) or left unmodified (WT group), whereas Arc-knockout cells remained unmodified as the Arc⁻/⁻ group. All groups were subsequently incubated with B16-F10 EVs/proteins to prepare vaccines for injection into WT recipient mice. (B) RT-qPCR analysis of mouse Arc (mArc) and mSynA expression in donor BM-DC/MΦs from the three groups, 24 h after exposure to B16-F10 EVs. Arc⁺ cells showed significantly elevated expression of both genes, whereas Arc⁻/⁻ cells showed reduced levels compared with WT controls. Mean ± SEM ( n = 3). One-way ANOVA; p-values shown. (C) RT-qPCR of immune-related and signaling genes (CD86, IFN-γ, IL-6, IL-10, TGF-β, VEGFa, VEGFR1, Notch) in primary BM-DC/MΦs stimulated with B16-F10 EVs. Arc⁺ DCs exhibited strong upregulation of immune activation genes and reduced TGF-β, whereas Arc⁻/⁻ DCs showed comparable levels of most markers besides an IL-10 elevation and VEGFR1 reduction. Mean ± SEM ( n = 3). One-way ANOVA; p-values shown. (D) Interaction assay between splenocytes from vaccinated mice and B16-F10 melanoma cells. CFSE-labeled splenocytes (red in i-iii; yellow in v) were co-incubated with NucSpot-labeled B16-F10 cells (purple in v). After 24 h, non-adherent cells were removed. WT and Arc⁺ splenocytes showed markedly increased tumor association, whereas Arc⁻/⁻ splenocytes did not. (E) Splenocytes isolated 8 days after melanoma vaccination were restimulated with B16-F10 EVs and analyzed by flow cytometry. Arc⁺ VLVs induced the strongest expansion of CD8⁺CD44⁺ and CD4⁺CD44⁺ T cells, whereas Arc −/− DEVs did not trigger a detectable response. Mean ± SEM ( n = 4-8). One-way ANOVA; p-values shown. (F) C57BL/6 mice received i.v. B16-F10-primed, DC2.4-derived DEVs or VLVs and were analyzed 72 h later in pooled lymph nodes (i) and the spleen (ii). Gating: singlets ◊ live ◊ CD45⁺ ◊ CD3⁺ ◊ CD4⁺/CD8⁺, with effector phenotype defined as CD62L lo CD44 hi . Arc⁺ VLVs significantly increased proliferation across CD3⁺, CD4⁺, CD8⁺, and effector subsets compared with B16⁻ DEV control. One-way ANOVA with Dunnett’s multiple comparisons; p-values shown. (G) Splenocytes isolated 12 days after vaccination were restimulated with B16-F10 EVs and analyzed for memory phenotypes. Representative plots show naïve (CD44⁻CD62L⁺), central memory (CD44⁺CD62L⁺), effector/effector memory (CD44⁺CD62L⁻), and pre-effector (CD44⁻CD62L⁻) subsets. Arc⁺ VLVs generated higher frequencies of CD8⁺ effector and central memory T cells. Data represents n = 4 mice per group. (H) Splenocytes isolated 12 days after vaccination were restimulated with B16-F10 or EO771 EVs. Arc⁺ VLVs induced significantly higher CD8⁺CD44⁺IFN-γ⁺ T-cell responses upon B16-F10 restimulation, with no response to EO771. CD4⁺CD44⁺IFN-γ⁺ cells expanded most strongly in the WT DEV group. Mean ± SEM ( n = 2-4). One-way ANOVA; significant p-values shown.

Article Snippet: DC2.4 murine dendritic cells (Millipore, SCC142), B16-F10 melanoma cells (ATCC, CRL-6475), and EO771 breast cancer cells (gift from the Weiss Lab, Cornell) were maintained in DMEM or RPMI-1640 (ATCC) supplemented with 10% FBS (Gibco) at 37 °C in 5% CO2.

Techniques: Transfection, Knock-Out, Incubation, Vaccines, Injection, Quantitative RT-PCR, Expressing, Activation Assay, Labeling, Isolation, Flow Cytometry, Derivative Assay, Control, Generated

Journal: bioRxiv

Article Title: Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

doi: 10.1101/2025.11.23.690059

Figure Lengend Snippet: (A) Schematic of the cytotoxicity assay. Splenocytes harvested on day 14 post-vaccination were labeled with CFSE and co-cultured with B16-F10 melanoma or EO771 breast cancer target cells pre-stained with CTFR at a total density of 0.6 × 10⁶ splenocytes per well in 100 µL (6 × 10⁶/mL), together with 3,000 tumor cells (bulk ratio ≈ 200:1). This high splenocyte density was selected to maintain ex vivo viability. Based on parallel phenotyping (≈ 3,000 IFN-γ⁺ CD8⁺ CD44⁺ cells per well), the effective effector-to-target ratio is ∼1:1 against 3,000 cancer cells. (B) EV flow cytometry of DC2.4-derived DEVs and VLVs generated at different B16-F10:DC2.4 cell ratios (0:1, 2:1, 1:1, 5:1). Histograms show CD40 signal on singlet CD9⁺CD45⁺CD11c⁺ vesicles (no-stain and dye-only controls indicated). Increasing the B16-F10:DC2.4 ratio shifts VLVs toward higher CD40 expression and increases CD40⁺ particle counts relative to DEVs. (C) Representative epifluorescence images of CTFR-labeled B16-F10 targets after 48 h co-culture with splenocytes from the indicated vaccine groups. Fewer red objects indicate greater tumor cell killing. Scale bars, 100 µm. (D) Ex vivo cytotoxicity at 48 h: live EO771 and B16-F10 target counts. Splenocytes from VLV-vaccinated mice significantly reduced B16-F10 cell numbers compared with PBS, B16⁺ DEV, or B16⁻ DEV controls, whereas EO771 targets showed no significant reductions. Symbols denote biological replicates ( n = 4-10); bars show mean ± SEM. One-way ANOVA with Dunnett’s multiple-comparison test vs. control; exact P values are shown when significant. (E) CTFR dilution profiles of live B16-F10 targets after 48 h co-culture. Peaks G0-G6 denote successive division generations defined by dye dilution (proliferation generations). VLV-vaccinated splenocytes reduce surviving targets across generations, yielding lower counts, especially in early generations. (F) Generation-resolved quantification of live B16-F10 targets at 48 h based on CTFR dilution. Bars show surviving cells in each division generation (G1-G6) after co-culture with splenocytes from the indicated groups (no-splenocyte and PBS controls, B16⁺/B16⁻ DEVs, B16⁺/B16⁻ VLVs). Mean ± SEM; n = 4-10 biological replicates. Two-way ANOVA with Dunnett’s multiple comparisons vs. PBS (or no-splenocyte for baseline); significant p-values annotated. (G) Splenocyte viability in co-culture (% live CD45⁺ cells) with or without mitogenic support (PHA-P). VLV-vaccinated splenocytes maintain viability comparable to controls across target types. Mean ± SEM; n = 2-4. (H) CFSE dilution of splenocytes after 48 h co-culture showing minimal proliferation (G0-G2 peaks) under assay conditions, indicating that target-cell loss primarily reflects cytotoxicity rather than splenocyte expansion. Unless noted otherwise, all assays used 0.6 × 10⁶ splenocytes and 3,000 targets per well in 100 µL (bulk ratio ≈ 200:1; estimated effective E:T ≈ 1:1 for B16-F10).

Article Snippet: DC2.4 murine dendritic cells (Millipore, SCC142), B16-F10 melanoma cells (ATCC, CRL-6475), and EO771 breast cancer cells (gift from the Weiss Lab, Cornell) were maintained in DMEM or RPMI-1640 (ATCC) supplemented with 10% FBS (Gibco) at 37 °C in 5% CO2.

Techniques: Cytotoxicity Assay, Labeling, Cell Culture, Staining, Ex Vivo, Flow Cytometry, Derivative Assay, Generated, Expressing, Co-Culture Assay, Comparison, Control

Journal: bioRxiv

Article Title: Repurposed endogenous virus-like vesicles mediate dendritic cell long-range antigen presentation and T cell activation for enhanced cancer vaccination

doi: 10.1101/2025.11.23.690059

Figure Lengend Snippet: (A) shows the timeline and dosage schedule of B16-F10 + DEV vaccines administered to wildtype C57BL6 mice, alongside the initial injection of melanoma cells and rechallenge with both melanoma and breast cancer cells. Tumor measurements were conducted using micro-CT scanning and double-blind caliper measurements by multiple independent researchers to ensure data precision. (B) Kaplan-Meier survival curves were monitored to day 132 (pre-rechallenge). Mice receiving B16 hi VLVs showed a substantial survival advantage (75% injection sites tumor-free and 60% mice alive at day 132; median not reached) versus DEV⁻ NC (0%; median 13.5 d), B16 lo DEV (33.3%; median 26.5 d), and B16 hi DEV (37.5%; median 22.5 d). Pairwise log-rank (Mantel-Cox) tests vs. DEV⁻ NC: B16 hi VLV, **P = 0.0011; B16 hi DEV, **P = 0.0015; B16 lo DEV, *P = 0.0116. All DEV⁻ mice were euthanized on days 13-17 per IACUC tumor-size limits. Each mouse received two flank injections. N: DEV⁻ (6 mice/12 tumors), B16 lo DEV (6/12), B16 hi DEV (8/16), B16 hi VLV (6/12). 3 independent experiments were performed with some animals pre-assigned for imaging and staining were not followed for survival. Median survival (d): DEV⁻ (13.5) / B16 lo DEV (26.5) / B16 hi DEV (22.5) / B16 hi VLV (>study limit). (C) Tumor growth curves from day 5 post-implantation. Arc⁺ (B16 hi VLV) tumors remained significantly smaller than controls. Per-site measurements (two flanks per mouse); mean ± SEM. N (sites): 12, 12, 16, 12 for DEV⁻, B16 lo DEV⁺, B16 hi DEV⁺, B16 hi VLV⁺, respectively (3 independent experiments). Statistics: two-way mixed-effects ANOVA (Dunnett’s multiple-comparisons); B16 lo DEV vs. B16 hi VLV: ****P < 0.0001. (D) When the final DEV⁻ control mouse reached humane endpoint on day 17, tumors were harvested for cross-sectional comparison. Left : representative tumors from one mouse per group (chosen a priori as the animal bearing the only or largest tumor for photographic comparison). Right: quantification of all injection sites at day 17 shows DEV⁻ > B16 lo DEV > B16 hi DEV > B16 hi Arc⁺ DEV > B16 hi VLV (mean ± SEM). Measurements were by caliper volume at the site level. n (sites): DEV⁻ = 12; B16 lo DEV⁺ = 12; B16 hi DEV⁺ = 16; B16 hi Arc⁺ DEV⁺ = 12; B16 hi VLV⁺ = 12. Statistics: two-way mixed-effects ANOVA with Dunnett’s multiple-comparisons test. **** P < 0.0001. (E) H&E: Arc⁺ VLV tumors were smaller with reduced viable tumor area, lower tumor-cell density, and less cohesive architecture compared with controls. CD8 IHC: A significant increase in CD8⁺ T cells was detected in B16 hi Arc⁺ DEV and VLV tumors vs. DEV⁻ controls. Notably, section-wide CD8⁺ counts in the VLV group appeared lower than Arc⁺ DEV, reflecting the scarcity of residual viable tumor in VLV sections (large areas comprised skin adnexa such as hair follicles and connective stroma). (F) Spleens were collected from the same tumor-bearing mice for IHC staining of CD4⁺ and CD8⁺ T cells. Splenic CD8⁺ T cell counts followed the order DEV⁻ < B16 lo DEV < B16 hi DEV = B16 hi Arc⁺ DEV, while the B16 hi Arc⁺ A5U⁺ VLV group displayed minimal CD8⁺ T cell staining. CD4⁺ T cells followed the order DEV⁻ < B16 lo DEV < B16 hi DEV < B16 hi Arc⁺ DEV = B16 hi Arc⁺ A5U⁺ VLV. (G) CD8⁺ cells in 5 representative tumor regions were quantified from CD8 IHC. Splenic CD4⁺ and CD8⁺ cells were counted in 5 representative regions. Mean ± SEM. Statistics: one-way ANOVA with Dunnett’s multiple-comparison, exact P values in panel. (H) Survival following challenge with melanoma cells and rechallenge with both melanoma and breast cancer cells: (i) In one of three independent experiments, long-term survivor mice were rechallenged with B16-F10 melanoma and EO771 breast cancer cells on day 132. A second batch of DEV⁻ controls was introduced as members of the first batch succumbed. (ii) Median survival times were as follows: 13.5 days for the first batch of DEV⁻ control (NC1); 14 days for the second batch of DEV⁻ control (NC2); 26.5 days for B16 lo DEV control; 22.5 days for B16 hi DEV control; and 154 days for the B16 hi VLV group. (I) Representative tumors collected at euthanasia from control and vaccinated mice. Each image shows all excised melanoma (B16-F10) and breast cancer (EO771) tumors from one representative experiment, arranged by group (DEV⁻ control vs. B16 hi DEV/VLV-vaccinated). (J) Quantification of tumor volumes at euthanasia. Control mice (DEV⁻) showed rapid melanoma progression and developed large tumors, leading to early mortality. In contrast, vaccinated groups (B16 hi DEV or B16 hi VLV) showed strong protection against melanoma, substantially extending the medium survival. This prolonged survival allowed the slower-growing breast cancer (EO771) tumors to reach larger sizes in some mice. (K) In addition to monitoring tumor growth, the behavioral well-being of treated mice was assessed, including locomotion and cognitive capacities using a novel object test. The movement tracks (orange) provide a visual representation of the paths taken by the mice within the test chamber. The heat map shows the density of movement within different areas, indicating the regions where the mice spent the most time. Besides the negative control mice, which are from new cancer cell injections, subjects in all experimental groups studied here are treated survivors after two rounds of tumor rechallenge. Representative tracks are shown here, with additional tracks available in the supplementary figure. (L) B16 hi vaccinated survivor mice exhibited increased average speed (i), total travel distance (ii), and number of entries into the novel object zone (iii), compared to control mice. The VLV group exhibited the highest level of locomotion activity. N = 3-5 in each sample group. Mean ± SEM. (M) MS label-free quantification enabled the identification and quantification of EV membrane proteins. The heat map provides a comparative analysis of protein expression levels across different sample groups. The heat map shows an upregulation of endogenous Envs in Ova-stimulated VLVs, compared to both the Ova ‒ DEV control and the Arc + /A5U ‒ DEV control. (N) Structural comparison of the MLV envelope (bottom) and AlphaFold prediction of ENV1 (top). MS analysis revealed a high degree of sequence similarity in the extracellular receptor-binding domain between MLV Env and a specific sequence enriched in Ova + VLVs (ENV1). A beta-strand sequence within this domain was identified as 100% conserved, highlighting significant structural conservation critical for receptor interaction.

Article Snippet: DC2.4 murine dendritic cells (Millipore, SCC142), B16-F10 melanoma cells (ATCC, CRL-6475), and EO771 breast cancer cells (gift from the Weiss Lab, Cornell) were maintained in DMEM or RPMI-1640 (ATCC) supplemented with 10% FBS (Gibco) at 37 °C in 5% CO2.

Techniques: Vaccines, Injection, Micro-CT, Imaging, Staining, Control, Comparison, Immunohistochemistry, Negative Control, Activity Assay, Quantitative Proteomics, Membrane, Expressing, Sequencing, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: Hsp47 drives obesity-associated breast cancer progression by enhancing asporin deposition in adipose tissue

doi: 10.1186/s13058-025-02076-9

Figure Lengend Snippet: Silence of Hsp47 in adipose tissue suppresses HFD-induced mammary tumor growth. ( A - B ) Tumor growth curve of EO771 xenografts in mammary glands of control and Hsp47 Adi-KO mice under HFD (A) conditions ( n = 9). ( C ) Tumor growth curve of Wnt xenografts in mammary glands of control and Hsp47 Adi-KO mice under HFD condition ( n = 4). ( D - F ) IHC staining and quantification of cell proliferation marker Ki67 and cell apoptosis marker active-Caspase3 in tumor tissue from EO771 xenograft models in control and Hsp47 Adi-KO mice under HFD condition ( n = 6). ( G - H ) SHG imaging and quantification of collagen fiber in tumor tissues from EO771 xenograft models in control and Hsp47 Adi-KO mice under HFD condition ( n = 4). ( I ) Administration of Col003 attenuated HFD-induced weight gain in mice ( n = 5). ( J ) Tumors and tumor growth curve of mouse EO771 orthotopic xenograft models in control and Col003 treated mice (HFD) ( n = 10).( K-M ) Quantification of cell proliferation marker Ki67, active-Caspase3, and collagen deposition in EO771 tumors in control and Col003 treated mice (HFD) ( n = 6).Results are presented as mean ± SEM; * p < 0.05; ** p < 0.01; independent Student’s t test. Scar bar: 100 μm

Article Snippet: Mouse breast cancer cell lines EO771 was obtained from ATCC.

Techniques: Control, Immunohistochemistry, Marker, Imaging

Journal: Breast Cancer Research : BCR

Article Title: Hsp47 drives obesity-associated breast cancer progression by enhancing asporin deposition in adipose tissue

doi: 10.1186/s13058-025-02076-9

Figure Lengend Snippet: Asporin mediates Hsp47 function in regulating obesity and obesity-associated tumor progression. ( A ) Kaplan-Meier survival analysis showing the association of asporin expression with tumor recurrence in basal-like breast cancer. ASPN: asporin( B ) The body weight growth curve of wide type and asporin KO mice (HFD) (WT group, n = 7; KO group, n = 8). ( C ) Tumor growth curve of mouse EO771 orthotopic xenograft models in control and asporin KO mice (HFD) ( n = 11). ( D - F ) IHC staining and quantification of cell proliferation marker Ki67, and active-Caspase3 inEO771 tumor tissue from control and asporin KO mice under HFD condition ( n = 6). ( G ) Masson’s Trichome staining images and quantification of collagen deposition in E0771 tumor tissues from control and asporin KO mice under HFD condition ( n = 3)( H ) The IVIS images showing treatment of asporin protein promoted breast cancer cell growth in the decellularized mammary gland from Hsp47 Adi-KO mice ( n = 3).Results are presented as mean ± SEM; * p < 0.05; ** p < 0.01; independent Student’s t test. Scar bar: 100 μm

Article Snippet: Mouse breast cancer cell lines EO771 was obtained from ATCC.

Techniques: Expressing, Control, Immunohistochemistry, Marker, Staining