Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: Vidu increases activation marker expression by tumor-specific CD8 + T cells. OT-1 splenocytes were cultured with either media (unstimulated) or E.G7-OVA cells (stimulated) for 1 hour, followed by no additional treatment (untreated) or the addition of Vidu and αQβ (treated). After 3 days of coculture, activation marker expression by OT-1 CD8 + T cells was analyzed by multicolor spectral flow cytometry. A, Representative histograms of CD25 expression on OT-1 CD8 + T cells. B, Frequency of CD25 + OT-1 CD8 + T cells and ( C ) MdFI of CD25 expression ( n = 8 mice/group) in untreated (black) and treated (red) samples. D, Representative histograms of PD-1 expression on OT-1 CD8 + T cells. E, Frequency of PD-1 + OT-1 CD8 + T cells and ( F ) MdFI of PD-1 expression ( n = 11 mice/group). G, Representative histograms of intracellular IFNγ expression by OT-1 CD8 + T cells. H, Frequency of IFNγ + OT-1 CD8 + T cells and ( I ) MdFI of IFNγ expression ( n = 11 mice/group). J, Representative histograms of intracellular TNFα expression by OT-1 CD8 + T cells. K, Frequency of TNFα + OT-1 CD8 + T cells and ( L ) MdFI of TNFα expression ( n = 6 mice/group). M, Representative histogram and dot plot and ( N ) frequency of polyfunctional IFNγ + TNFα + OT-1 CD8 + T cells ( n = 7 mice/group). Each symbol connected by a line represents an individual mouse in untreated and Vidu/αQβ conditions and the mean of the group, with error bars indicating the SEM. Statistical significance was determined using a paired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Activation Assay, Marker, Expressing, Cell Culture, Flow Cytometry

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: Vidu decreases proliferation of tumor-specific CD8 + T cells while enhancing activation of cells that divided. OT-1 splenocytes were labeled with a proliferation tracking dye (CellTrace Violet) and cultured with either media (unstimulated) or E.G7-OVA cells (stimulated) for 1 hour, followed by no additional treatment (untreated) or the addition of Vidu and αQβ (treated). After 3 days of coculture, proliferation and activation marker expression by OT-1 CD8 + T cells was analyzed by multicolor spectral flow cytometry. A, Percent divided and ( B ) proliferation index of OT-1 CD8 + T cells ( n = 10 mice/group). C, Representative histograms showing OT-1 CD8 + T-cell proliferation. D, MdFI of IFNγ expression by undivided and divided OT-1 CD8 + T cells ( n = 10 mice/group) and ( E ) representative dot plot and histogram in unstimulated (blue), stimulated/untreated (black), and stimulated/treated (red) samples. F, MdFI of TNFα expression by undivided and divided OT-1 CD8 + T cells ( n = 4 mice/group) and ( G ) representative dot plot and histogram. H, MdFI of PD-1 expression by undivided and divided OT-1 CD8 + T cells ( n = 10 mice/group) and ( I ) representative dot plot and histogram. J, MdFI of CD25 expression by undivided and divided OT-1 CD8 + T cells ( n = 7 mice/group) and ( K ) representative dot plot and histogram. E , G , I , and K, Red arrows indicate undivided population, and vertical dashed lines indicate separation of negative (left) from positive (right) marker expression. Each symbol connected by a line represents an individual mouse in untreated and Vidu/αQβ conditions and the mean of the group, with error bars indicating the SEM. Statistical significance was determined using a paired t test ( A and B ) or two-way ANOVA with Sidak multiple comparisons test ( D–J ): *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant.

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Activation Assay, Labeling, Cell Culture, Marker, Expressing, Flow Cytometry

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: Vidu modestly increases granzyme B expression by tumor-specific CD8 + T cells but has limited impact on cytotoxicity. OT-1 splenocytes were cultured with E.G7-OVA cells or SIINFEKL peptide (stimulated) for 1 hour, followed by no additional treatment (untreated) or the addition of Vidu and αQβ (treated). After 3 days of coculture, granzyme B expression or cytotoxicity by OT-1 CD8 + T cells was analyzed. A, Frequency of granzyme B + OT-1 CD8 + T cells and MdFI of granzyme B expression ( n = 5 mice/group) in untreated (black) and treated (red) samples. B, Secreted granzyme B as determined by ELISA ( n = 3 mice/group). C, treatment and cytotoxicity schema of ( D and E ) isolated CD8 + T cells were cocultured with target E.G7-OVA tumor cells at various effector:target ratios for 18 hours. D, Percent cytotoxicity of OT-1 CD8 + T cells and ( E ) viable E.G7-OVA cells/mL was determined ( n = 3–4 mice/group). SIINFEKL peptide was used at a final concentration of 10 ng/mL. Each symbol connected by a line represents an individual mouse in untreated and Vidu/αQβ conditions and the mean of the group, with error bars indicating the SEM. Statistical significance was determined using a paired t test: *, P < 0.05; **, P < 0.01; ns, not significant. [ C, Created in BioRender. Weiner, G. (2026) https://BioRender.com/s3vjcvy .]

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: Vidu enhances triple-positive tumor-specific CD8 + T-cell activation marker expression after culture with target cells for 3 days. OT-1 splenocytes were cultured with either media (unstimulated) or E.G7-OVA cells (stimulated) for 1 hour, followed by no additional treatment (untreated) or the addition of Vidu and αQβ (treated). After 3 days of coculture, activation and exhaustion marker expression by OT-1 CD8 + T cells was analyzed by multicolor spectral flow cytometry. A, Representative dot plot and histogram in unstimulated (blue), stimulated/untreated (black), and stimulated/treated (red) samples showing the gating strategy of PD-1 + OT-1 CD8 + T cells and their coexpression of LAG3 ± TIM3. B, Double- and triple-positive OT-1 CD8 + T-cell populations were analyzed for activation marker expression in untreated (black) and treated (red) samples ( n = 8 mice/group). C, Frequency of CD25 + OT-1 CD8 + T cells and ( D ) MdFI of CD25 expression ( n = 8 mice/group). E, Frequency of IFNγ + OT-1 CD8 + T cells and ( F ) MdFI of IFNγ expression ( n = 8 mice/group). G, Frequency of IFNγ + TNFα + OT-1 CD8 + T cells ( n = 5 mice/group). Each symbol connected by a line represents an individual mouse in untreated and Vidu/αQβ conditions and the mean of the group, with error bars indicating the SEM. Statistical significance was determined using a two-way ANOVA with Sidak multiple comparisons test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Activation Assay, Marker, Expressing, Cell Culture, Flow Cytometry

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: Vidu has complex effects on the expression of markers of activation and exhaustion after long-term in vitro culture. A, OT-1 splenocytes were cultured with SIINFEKL peptide (stimulated) for 1 hour, followed by no additional treatment (untreated) or the addition of Vidu and αQβ (treated). Additional SIINFEKL stimulation on days 1–4 for a total of 5 SIINFEKL doses was provided to evaluate chronic antigen stimulation on OT-1 CD8 + T cells. After 7 and 14 days of culture, activation and exhaustion marker expression by OT-1 CD8 + T cells was analyzed by multicolor spectral flow cytometry. Media were changed every 3 days with supplemental IL7 and IL15. Exhaustion status of OT-1 CD8 + T cells was determined by the ( B ) frequency of triple-positive OT-1 CD8 + T cells and ( C ) MdFI of PD-1 on OT-1 CD8 + T cells ( n = 4–5 mice/group) in untreated (black) and treated (red) samples. Activation status of OT-1 CD8 + T cells was determined by the ( D ) frequency of CD25 + OT-1 CD8 + T cells and ( E ) MdFI of CD25 expression ( n = 4 mice/group). CD8 + ( F ) MdFI of IFNγ, ( G ) frequency of IFNγ + TNFα + , and ( H ) MdFI of granzyme B expressed by OT-1 CD8 + T cells ( n = 4 mice/group). Each symbol connected by a line represents an individual mouse in untreated and Vidu/αQβ conditions and the mean of the group, with error bars indicating the SEM. Statistical significance was determined using a two-way ANOVA with Sidak multiple comparisons test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. [ A, Created in BioRender. Weiner, G. (2026) https://BioRender.com/s3vjcvy .]

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Expressing, Activation Assay, In Vitro, Cell Culture, Marker, Flow Cytometry

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: IT Vidu injections enhance tumor regression and markedly increase the number of circulating tumor-specific T cells. A, In vivo treatment schema evaluating the impact of IT Vidu treatment. CD8 + CD8 + . B, Kaplan–Meyer curve of saline (untreated, black) vs. Vidu (treated, red) mouse survival ( n = 7–8 mice/group). C, Spider plots of saline (left) vs. Vidu (right) treated mice showing individual tumor volumes over time; dashed vertical lines indicate day of IT injection, “X” indicates the time point an individual mouse was sacrificed, and a filled circle indicates a mouse surviving to the endpoint of the study ( n = 7–8 mice/group). D, Frequency of IT tumor-specific CD8 + T cells, as indicated by tetramer positivity, out of total CD8 + T cells ( n = 3–7 mice/group). E, Frequency of IT PD-1 + OT-1 CD8 + T cells and ( F ) MdFI of PD-1 expression ( n = 2–6 mice/group). G, Frequency of IT PD-1 + LAG3 + TIM3 + tumor-specific CD8 + T cells ( n = 2–6 mice/group). H, Frequency of circulating tumor-specific CD8 + T cells out of total CD8 + T cells ( n = 3–8 mice/group). I, Frequency of circulating PD-1 + OT-1 CD8 + T cells and ( J ) MdFI of PD-1 expression ( n = 3–7 mice/group). Each symbol represents an individual mouse and the mean of the group, with error bars indicating the SD. Statistical significance was determined using a ( B ) log-rank (Mantel–Cox) test for survival, ( D–H ) two-way ANOVA with Sidak multiple comparisons test and ( I and J ) unpaired t test: *, P < 0.05; ***, P < 0.001; ns, not significant. [ A, Created in BioRender. Weiner, G. (2026) https://BioRender.com/s3vjcvy .]

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: In Vivo, Saline, Injection, Expressing

Journal: Cancer Research Communications

Article Title: Intratumoral Virus-Like Particles Containing a TLR9 Agonist Combined with Systemic αPD-1 Activate Tumor-Specific CD8 + T Cells

doi: 10.1158/2767-9764.CRC-26-0175

Figure Lengend Snippet: αPD-1 enhanced the antitumor activity of Vidu and sustained IT T cells. A, In vivo treatment schema evaluating the impact of combination αPD-1 and Vidu CD8 + CD8 + . B, Tumor volumes over time; arrows indicate day of tumor implantation and treatment ( n = 4–10 mice/group). C, Frequency of IT tumor-specific CD8 + T cells, as indicated by tetramer positivity, out of total CD8 + T cells. Frequency of tumor-specific CD8 + T cells out of total CD8 + T cells in ( D ) circulation, ( E ) DLN, and ( F ) spleen. Frequency of IT ( G ) CD4 + T cells out of single cells. H, Frequency of IT CXCR3 + CD4 + T cells out of total CD4 + T cells ( n = 3–5 mice/group). Each symbol represents an individual mouse and the mean of the group with SD. Statistical significance was determined using a ( B–H ) one-way ANOVA with Tukey test for multiple comparisons and ( C–H ) unpaired t test between Vidu and combination groups: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. [ A, Created in BioRender. Weiner, G. (2026) https://BioRender.com/s3vjcvy .]

Article Snippet: After 3 days of culture, untouched CD8 + T cells were isolated for use as effector cells using a CD8α + T-cell isolation kit (Miltenyi, cat. #130-104-075).

Techniques: Activity Assay, In Vivo, Tumor Implantation

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: scRNA-seq reveals differential immune infiltration in αOX40-treated tumors based on response (A) Schematic of the bilateral MC38 tumor model assessing αOX40 response. Humanized OX40 mice received three doses of αOX40, followed by resection of the left tumor for scRNA-seq and flow cytometry analysis. Contralateral tumor dynamics and survival were monitored longitudinally. (B) UMAP visualization of scRNA-seq data from immune cells in MC38-bearing mice following αOX40 treatment. Cells are color-coded by annotated cell type. (C) Heatmap depicting nine transcriptionally distinct immune cell subpopulations. (D) Pie chart shows the relative abundance of nine immune cell clusters in αOX40 responders and nonresponders. (E) Flow cytometry analysis of tumor-infiltrating immune cell frequencies in αOX40-treated MC38-bearing mice. Frequencies of CD4 + T cells, CD8 + T cells, and macrophages were quantified after the third αOX40 dose (control, n = 5 mice; mice with a robust therapeutic response named as responder, n = 4 mice; mice with minimal to no response named as nonresponder, n = 4 mice). Data represent mean ± SD from one of two independent experiments (E). Statistical significance was determined using one-way ANOVA with multiple comparisons. ∗∗ p < 0.01.

Article Snippet: CD8 + T cells were magnetically isolated from spleens (after four treatments) and tumor tissues (after three treatments) of Control-, OX40-, MPLA+IFNγ-, or Combo-treated mice using CD8α (Ly-2) MicroBeads (130-117-044, Miltenyi Biotec) and co-cultured with CFSE-labeled MC38 target cells (1×10 5 cells/well) at an effector-to-target ratio of 10:1 in complete RPMI-1640 medium supplemented with 10% FBS for 36 h at 37°C.

Techniques: Flow Cytometry, Control, Clinical Proteomics

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: CD8 + T cells were magnetically isolated from spleens (after four treatments) and tumor tissues (after three treatments) of Control-, OX40-, MPLA+IFNγ-, or Combo-treated mice using CD8α (Ly-2) MicroBeads (130-117-044, Miltenyi Biotec) and co-cultured with CFSE-labeled MC38 target cells (1×10 5 cells/well) at an effector-to-target ratio of 10:1 in complete RPMI-1640 medium supplemented with 10% FBS for 36 h at 37°C.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: The antitumor efficacy of the Combo therapy is contingent upon CD8 + T cells and macrophages (A) UMAP of scRNA-seq data from tumor-infiltrating immune cells in OX40-humanized MC38-bearing mice treated with MPLA, IFN-γ, αOX40, or Combo. Cells are color-coded by annotated cell type. (B) Bubble chart showing the top variable marker genes for identified immune cell types. (C) Pie chart shows the relative abundance of 11 immune cell clusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) Macrophage frequency and absolute count in tumors of MC38-bearing mice after two and three treatment cycles with MPLA, IFN-γ, αOX40, or Combo, analyzed by flow cytometry ( n = 5 mice per group). (E) Schematic of CD8 + T cell depletion assay. (F) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study of CD8 + T cell ( n = 6 mice per group). (G) Schematic of macrophage depletion assay in early and late stage. (H and I) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study in (G) ( n = 6–10 mice per group). Data are shown as means ± SD from one of two independent experiments (D, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (D). Log rank test was used (F, H, and I) for statistical comparison. n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: CD8 + T cells were magnetically isolated from spleens (after four treatments) and tumor tissues (after three treatments) of Control-, OX40-, MPLA+IFNγ-, or Combo-treated mice using CD8α (Ly-2) MicroBeads (130-117-044, Miltenyi Biotec) and co-cultured with CFSE-labeled MC38 target cells (1×10 5 cells/well) at an effector-to-target ratio of 10:1 in complete RPMI-1640 medium supplemented with 10% FBS for 36 h at 37°C.

Techniques: Marker, Control, Flow Cytometry, Depletion Assay, Comparison

Journal: bioRxiv

Article Title: Serial Thermal Ablation Induces Abscopal Antitumor Immunity and Reveals Targetable CSF1R-Dependent Resistance in Pancreatic Cancer

doi: 10.64898/2026.04.05.713683

Figure Lengend Snippet: A) Representative 20x NK1.1 immunohistochemistry staining images. Scale bars 100 µm. B) NK1.1 staining is significantly increased in 3 RFA contralateral tumors compared to 1 RFA contralateral tumors (***p<0.0001). NK1.1 is significantly increased in 3 RFA-treated tumors compared to 3 RFA contralateral tumors (*p=0.0141). n=10 fields analyzed per tumor. C) Representative 20x immunofluorescence images of CD4 (purple), CD8α (red), GZMB (green), DAPI nuclear stain (blue). D) CD4 + GZMB + cells are not increased in 3 RFA contralateral tumors. E) CD8α + GZMB + cells are significantly increased in the three RFA contralateral tumors compared to 1 RFA contralateral (****p<0.0001) and 3 RFA RFA-treated tumors (**p=0.0046). n=8 fields analyzed per group. F) Schematic of the protocol to evaluate the effects of CD8 depletion on serial RFA-treated and contralateral tumors. Created using BioRender . G) CD8α depletion significantly reduces the %CD8α + cells per live cells in RFA-treated tumors. H) α-CD8α significantly increases the tumor volume of RFA-treated (*p<0.05; ***p<0.001) and I) contralateral tumors after 2 RFA treatments but does not significantly increase the final tumor volumes after the third RFA treatment. Statistics were done using Prism GraphPad software.

Article Snippet: The following primary antibodies were used: CD4 (1:35, ab288724, abcam), CD8α (1:50, MAB116-100, R&D Systems), GZMB (1:100, AF1865, R&D Systems).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Serial Thermal Ablation Induces Abscopal Antitumor Immunity and Reveals Targetable CSF1R-Dependent Resistance in Pancreatic Cancer

doi: 10.64898/2026.04.05.713683

Figure Lengend Snippet: A) Experimental design setup. B) Serial thermal ablation using RFA in combination with anti-PD-L1 and Quemli does not significantly reduce the volume of treated tumors. C) However, serial RFA in combination with anti-PD-L1 and Quemli significantly reduces the tumor volume of contralateral tumors compared to serial RFA + IgG+Vehicle (*p<0.05) and compared to serial RFA + Quemli treated alone (****p<0.0001). D) Representative 20x CSF1R immunohistochemistry images. E) Quantification of CSF1R + cells showed an increase in CSF1R + area per field in serial RFA tumors + anti-PD-L1 with or without Quemli compared to serial RFA + vehicle in both RFA-treated and F) contralateral tumors. A two-way Anova was used in Prism GraphPad for statistical comparisons. G) Experimental design setup. H) Serial thermal ablation using RFA in combination with CSF1R inhibition, anti-PD-L1 and Quemli significantly reduces the volume of ablated tumors (**p<0.01; ***p<0.001) and I) contralateral tumors (**p<0.01; ***p<0.001). J) Combination treatment using 3 RFA + anti-PD-L1, Quemli and CSF1R inhibition significantly increases the infiltration of CD8 + T cells in treated (**p<0.01; *p<0.05) and K) contralateral tumors (**p<0.01; *p<0.05) . L) Combination treatment using 3 RFA + anti-PD-L1, Quemli and CSF1R inhibition significantly increases GZMB staining in treated (***p<0.001; **p<0.01) and M) contralateral tumors (*p<0.05; **p<0.01; ***p<0.001). A two-way ANOVA in Prism GraphPad was used for statistical analysis. Scale bars 50 µm.

Article Snippet: The following primary antibodies were used: CD4 (1:35, ab288724, abcam), CD8α (1:50, MAB116-100, R&D Systems), GZMB (1:100, AF1865, R&D Systems).

Techniques: Immunohistochemistry, Inhibition, Staining

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A to I ) Serpine1 +/+ and Serpine1 −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected at day 21. [(A) and (B)] Flow cytometry quantification of frequencies of total T cells (CD45 + CD3 + ) (A) and conventional dendritic cells (CD45 + F4/80 − CD11c + MHCII + ) (B) among live cells in orthotopic tumors. (C) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). (D) Quantitative RT-PCR analysis of Arg1 in BMDMs treated with vehicle or recombinant PAI1 (rPAI1) for 20 hours. Each dot represents individual primary BMDM lines. (E) Flow cytometry quantification of frequency of CD8 + T cells (CD45 + CD3 + CD8 + ) among live cells in orthotopic tumors. [(F) and (G)] Flow cytometry quantification of frequencies and absolute numbers of GZMB + CD8 + T cells (F) and Ki67 + CD8 + T cells (G) following a 5-hour ex vivo PMA/ionomycin stimulation of tumor digests. (H) Co-IF staining for GZMB (green), CD8 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). (I) Co-IF staining for Ki67 (red), CD8 (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( J ) Experimental design for CD8 + T cell depletion. Mice received control immunoglobulin G (IgG) or anti-CD8 antibody twice per week, starting 5 days postimplantation (7940B). Tumors were harvested at day 21. ( K ) Tumor weight of orthotopic tumors with CD8⁺ T cell antibody-mediated depletion. Symbols in (A) to (I) and (K) represent individual mice. Data are means ± SEM. P values were determined by two-tailed unpaired t test [(A), (B), right of (C), (D), (E) to (G), and right of (H)], Mann-Whitney test [left of (C), left of (H), and (I)], and Mann-Whitney test with Holm-Sidak post hoc (K).

Article Snippet: Depletion of CD8 + T cells were performed starting from 5 days following orthotopic injection of 7940B cells by administration of intraperitoneal injections of 200 μg of anti-mouse CD8α mAb (2.43, no. BE0061, BioXCell) or control isotype antibody (LTF-2, no. BE0090, BioXCell) twice per week for the entire course of the experiments.

Techniques: Flow Cytometry, Staining, Quantitative RT-PCR, Recombinant, Ex Vivo, Control, Two Tailed Test, MANN-WHITNEY

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. Plat +/+ and Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from Plat +/+ and Plat −/− mice. ( C ) ELISA of tPA in tissue supernatants from pancreata of Plat +/+ mice (no tumor) and orthotopic tumors from Plat +/+ and Plat −/− mice. ( D ) Picrosirius red staining of orthotopic tumors, and corresponding quantification ( n = 5 FOV per animal). ( E ) Co-IF staining for cCasp3 (red), ECAD (green), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 4 FOV per animal). ( F to H ) Flow cytometry quantification of frequencies of total T cells (F), CD8 + T cells (G), and conventional dendritic cells (H) among live cells in orthotopic tumors. ( I ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (I) represent individual mice. Data are means ± SEM. P values were determined by Mann-Whitney test [(B) and (F)], two-way ANOVA with Holm-Sidak post hoc (C), and two-tailed unpaired t test [(D), (E), and (G) to (I)].

Article Snippet: Depletion of CD8 + T cells were performed starting from 5 days following orthotopic injection of 7940B cells by administration of intraperitoneal injections of 200 μg of anti-mouse CD8α mAb (2.43, no. BE0061, BioXCell) or control isotype antibody (LTF-2, no. BE0090, BioXCell) twice per week for the entire course of the experiments.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: A stromal PAI1-tPA axis orchestrates immunosuppression in pancreatic cancer

doi: 10.1126/sciadv.aea6734

Figure Lengend Snippet: ( A ) Experimental outline of murine PDAC model. WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice were orthotopically implanted with pancreatic cancer cells (7940B), and tumors were collected 21 days postimplantation. ( B ) Tumor weight of orthotopic tumors harvested from WT, Serpine1 −/− , and Serpine1 −/− ; Plat −/− mice. ( C ) IHC staining for CD8 in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). ( D ) Co-IF staining for Arg1 (green), F4/80 (red), and DAPI (blue) in orthotopic tumors, and corresponding quantification ( n = 3 FOV per animal). Symbols in (B) to (D) represent individual mice. Data are means ± SEM. P values were determined by one-way ANOVA with Holm-Sidak post hoc [(B) to (D)]. ( E ) Working model. Left: Hypoxia stabilizes HIF proteins, inducing PAI1 expression in pancreatic CAFs. Although tPA levels are elevated in PDAC, PAI1 predominantly inhibits its activity, thereby suppressing antitumor immunity. Middle: Elimination of stromal PAI1 restores tPA activity, enhancing antitumor CD8 + T cell responses, accompanied by alleviation of immunosuppressive TAM phenotypes and increased dendritic cell (DC) infiltration. Stromal PAI1-driven tumor growth depends on CD8 + T cells. Right: Removal of stromal tPA abolishes residual tPA activity, further promoting an immunosuppressive TME and tumor growth.

Article Snippet: Depletion of CD8 + T cells were performed starting from 5 days following orthotopic injection of 7940B cells by administration of intraperitoneal injections of 200 μg of anti-mouse CD8α mAb (2.43, no. BE0061, BioXCell) or control isotype antibody (LTF-2, no. BE0090, BioXCell) twice per week for the entire course of the experiments.

Techniques: Immunohistochemistry, Staining, Expressing, Activity Assay