Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. P14 CD8 T cells (5 × 10 6 cells, CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ), and their kinetics were monitored in the blood. (B) Kinetics of adoptively transferred P14 CD8 T cells (CD45.1 + DbGP33 tetramer + CD8 T cells) in the blood without infection. The frequency of P14 CD8 T cells at 1.5 days post-transfer was set as 100%. Each symbol represents the mean and error bars indicate SEM. (C) CD44, CD62L, and CD127 expression on splenic P14 CD8 T cells before transfer and at 35 days post-transfer. As a control, the expression of these markers on DbGP33 + memory CD8 T cells obtained from the spleen of LCMV-Arm infected mice (> day 100 post-infection) is shown. Histograms were gated on DbGP33 + CD8 T cells. (D) Experimental design for panels E to G. P14 CD8 T cells (long-lived P14, CD45.1 +/+ ), housed for 36 days after transfer in B6 mice, were mixed with an equal number of freshly isolated P14 CD8 T cells (fresh P14, CD45.1 + CD45.2 + ). The mixture (1 × 10 3 P14 CD8 T cells of each population) was adoptively transferred into CD45.2 +/+ B6 mice, followed by LCMV-Arm infection. (E and F) Percentages of progeny derived from fresh and long-lived P14 CD8 T cells within total P14 CD8 T cells (E) and their kinetics (F) in the blood post-infection. (G) The number of memory precursor effector P14 cells (CD127 + KLRG1 − ) in the blood on day 8 post-infection. (H and I) Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, housed for 1.5 days (short-housed P14) and 35 days (long-lived P14), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. Percentages of progeny derived from each population within total P14 CD8 T cells (H) and their kinetics (I) in the blood post-infection. (J and K) Responses of long-lived P14 cells during chronic LCMV-Clone 13 infection. Long-lived P14 CD8 T cells (1 × 10 3 cells, CD45.1 +/+ ), housed for 74 days in B6 mice, were adoptively co-transferred with an equal number of fresh P14 CD8 T cells (1 × 10 3 cells, CD45.1 + CD45.2 + ) into B6 mice (CD45.2 +/+ ), followed by LCMV-Clone 13 infection. Percentages of progeny derived from each population within total P14 CD8 T cells (J) and their kinetics (K) in the blood post-infection. Data are representative of 2 or more independent experiments with 3 or more mice per group. In E–K, each symbol represents an individual mouse, and lines indicate paired comparisons within the same mice. Statistical analysis was performed using paired t test (G) and one-way ANOVA (E, F, H, I, J, and K). ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Infection, Expressing, Control, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B–F. P14 CD8 T cells (CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ). On 1.5 days (fresh) and 35 days (long-lived) post-transfer, P14 CD8 T cells (CD45.1 + DbGP33 + CD8 T cells) were sorted from the spleens of recipient mice ( n = 4 each) for RNA-seq analysis. Gene expression (read counts) was analyzed using DESeq2. (B) Heatmap of sample-to-sample distances using the Poisson Distance. (C) Fold changes and adjusted p -values of gene expression in long-lived P14 CD8 T cells relative to fresh P14 CD8 T cells. (D–F) Normalized read counts of Il18r1 (D), Cxcr3 (E), and Nt5e (F) in long-lived and fresh P14 CD8 T cells. Each symbol represents an individual mouse. (G–K) Protein expression levels of IL-18Rα, CXCR3, and CD73 on long-lived (>30 days post-transfer) and fresh P14 CD8 T cells in the spleens. (G) Representative histograms were gated on DbGP33 + P14 CD8 T cells. (H to J) Data were pooled from 6 independent experiments, and each symbol represents an individual mouse. (K) Heterogeneity of naïve P14 CD8 T cells (gated on DbGP33 + P14 CD8 T cells) based on the expression of IL-18Rα, CXCR3, and CD73. (L) Heterogeneity of endogenous bulk and DbGP33 + naïve CD8 T cells. Tetramer enrichment was performed using the cells isolated from the spleen and LNs (inguinal and brachial LNs) of naïve B6 mice, and the expression of IL-18Rα, CXCR3, and CD73 on either DbGP33 + or bulk CD44 Lo CD8 T cells was analyzed. Statistical analyses were performed using Walt’s test (D–F) and unpaired t test (H–J). *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: RNA Sequencing, Gene Expression, Expressing, Isolation

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B and C. Congenically distinct B6 mice were conjoined by parabiosis to effectively transfer donor CD8 T cells into another mouse. Parabiotic mice were separated 5 weeks after conjoining, which allowed for the prolonged monitoring of donor-derived CD8 T cell phenotype. 1.5 to 2.5 months after fusion reversal, the phenotype of donor- and host-derived bulk CD44 Lo (B) and DbGP33 + CD44 Lo (C) CD8 T cells in the spleen was analyzed using tetramer enrichment. Data are pooled from 3 independent experiments with 10 mice. Each symbol represents an individual mouse and lines indicate paired comparisons within the same mouse. Statistical analysis was performed using paired t test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. Five naïve CD8 T cell subsets were sorted from the spleen of P14 TCR transgenic mice. Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, including each sorted subset and freshly isolated P14 CD8 T cells (control), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. The responses of sorted and control P14 populations in the blood were monitored over time. (B) Kinetics of the frequency of sorted and control P14 CD8 T cells. The frequency of control P14 cells at day 8 post-infection was set as 100. Each symbol represents the mean, and error bars indicate the SEM. (C) Experimental design for panels D and E. IL18Rα Lo CD73 Lo (DN: double negative) or IL18Rα Hi CD73 Hi (DP: double positive) CD44 Lo naïve CD8 T cells were sorted from splenocytes of B6 mice (CD45.2 +/+ ). The number of DbGP33 tetramer + CD8 T cells was confirmed using a portion of the sorted cells via tetramer enrichment. Bulk sorted cells, containing equal numbers of DbGP33 tetramer + cells, were separately transferred into recipient mice (CD45.1 + CD45.2 + ), followed by LCMV-Arm infection. On days 7–8 post-infection, tetramer enrichment was performed to analyze the response of donor-derived DbGP33 + CD8 T cells in the spleen. (D) Representative flow plots. (E) Frequency of donor-derived DbGP33 + CD8 T cells among total DbGP33 + CD8 T cells in the spleen. Each symbol represents an individual mouse. Data were pooled from 2 independent experiments with 5 or 9 mice per group. Statistical analyses were performed using one-way ANOVA (B) and unpaired t test (E). * p < 0.05; ** p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques: Transgenic Assay, Isolation, Control, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: IL-18Rα Hi CD73 Hi CD44 Lo and IL-18Rα Lo CD73 Lo CD44 Lo CD8 T cells were sorted from splenocytes of naïve B6 mice.

Article Snippet: The following antibodies were purchased from Miltenyi Biotec: CD44 (clone: REA664) and Ly6C (clone: REA796).

Techniques:

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. P14 CD8 T cells (5 × 10 6 cells, CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ), and their kinetics were monitored in the blood. (B) Kinetics of adoptively transferred P14 CD8 T cells (CD45.1 + DbGP33 tetramer + CD8 T cells) in the blood without infection. The frequency of P14 CD8 T cells at 1.5 days post-transfer was set as 100%. Each symbol represents the mean and error bars indicate SEM. (C) CD44, CD62L, and CD127 expression on splenic P14 CD8 T cells before transfer and at 35 days post-transfer. As a control, the expression of these markers on DbGP33 + memory CD8 T cells obtained from the spleen of LCMV-Arm infected mice (> day 100 post-infection) is shown. Histograms were gated on DbGP33 + CD8 T cells. (D) Experimental design for panels E to G. P14 CD8 T cells (long-lived P14, CD45.1 +/+ ), housed for 36 days after transfer in B6 mice, were mixed with an equal number of freshly isolated P14 CD8 T cells (fresh P14, CD45.1 + CD45.2 + ). The mixture (1 × 10 3 P14 CD8 T cells of each population) was adoptively transferred into CD45.2 +/+ B6 mice, followed by LCMV-Arm infection. (E and F) Percentages of progeny derived from fresh and long-lived P14 CD8 T cells within total P14 CD8 T cells (E) and their kinetics (F) in the blood post-infection. (G) The number of memory precursor effector P14 cells (CD127 + KLRG1 − ) in the blood on day 8 post-infection. (H and I) Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, housed for 1.5 days (short-housed P14) and 35 days (long-lived P14), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. Percentages of progeny derived from each population within total P14 CD8 T cells (H) and their kinetics (I) in the blood post-infection. (J and K) Responses of long-lived P14 cells during chronic LCMV-Clone 13 infection. Long-lived P14 CD8 T cells (1 × 10 3 cells, CD45.1 +/+ ), housed for 74 days in B6 mice, were adoptively co-transferred with an equal number of fresh P14 CD8 T cells (1 × 10 3 cells, CD45.1 + CD45.2 + ) into B6 mice (CD45.2 +/+ ), followed by LCMV-Clone 13 infection. Percentages of progeny derived from each population within total P14 CD8 T cells (J) and their kinetics (K) in the blood post-infection. Data are representative of 2 or more independent experiments with 3 or more mice per group. In E–K, each symbol represents an individual mouse, and lines indicate paired comparisons within the same mice. Statistical analysis was performed using paired t test (G) and one-way ANOVA (E, F, H, I, J, and K). ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti- CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti- CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Infection, Expressing, Control, Isolation, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B–F. P14 CD8 T cells (CD45.1 + ) were adoptively transferred into B6 mice (CD45.2 + ). On 1.5 days (fresh) and 35 days (long-lived) post-transfer, P14 CD8 T cells (CD45.1 + DbGP33 + CD8 T cells) were sorted from the spleens of recipient mice ( n = 4 each) for RNA-seq analysis. Gene expression (read counts) was analyzed using DESeq2. (B) Heatmap of sample-to-sample distances using the Poisson Distance. (C) Fold changes and adjusted p -values of gene expression in long-lived P14 CD8 T cells relative to fresh P14 CD8 T cells. (D–F) Normalized read counts of Il18r1 (D), Cxcr3 (E), and Nt5e (F) in long-lived and fresh P14 CD8 T cells. Each symbol represents an individual mouse. (G–K) Protein expression levels of IL-18Rα, CXCR3, and CD73 on long-lived (>30 days post-transfer) and fresh P14 CD8 T cells in the spleens. (G) Representative histograms were gated on DbGP33 + P14 CD8 T cells. (H to J) Data were pooled from 6 independent experiments, and each symbol represents an individual mouse. (K) Heterogeneity of naïve P14 CD8 T cells (gated on DbGP33 + P14 CD8 T cells) based on the expression of IL-18Rα, CXCR3, and CD73. (L) Heterogeneity of endogenous bulk and DbGP33 + naïve CD8 T cells. Tetramer enrichment was performed using the cells isolated from the spleen and LNs (inguinal and brachial LNs) of naïve B6 mice, and the expression of IL-18Rα, CXCR3, and CD73 on either DbGP33 + or bulk CD44 Lo CD8 T cells was analyzed. Statistical analyses were performed using Walt’s test (D–F) and unpaired t test (H–J). *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti- CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti- CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: RNA Sequencing, Gene Expression, Expressing, Isolation

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panels B and C. Congenically distinct B6 mice were conjoined by parabiosis to effectively transfer donor CD8 T cells into another mouse. Parabiotic mice were separated 5 weeks after conjoining, which allowed for the prolonged monitoring of donor-derived CD8 T cell phenotype. 1.5 to 2.5 months after fusion reversal, the phenotype of donor- and host-derived bulk CD44 Lo (B) and DbGP33 + CD44 Lo (C) CD8 T cells in the spleen was analyzed using tetramer enrichment. Data are pooled from 3 independent experiments with 10 mice. Each symbol represents an individual mouse and lines indicate paired comparisons within the same mouse. Statistical analysis was performed using paired t test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To prevent anti- CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti- CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: (A) Experimental design for panel B. Five naïve CD8 T cell subsets were sorted from the spleen of P14 TCR transgenic mice. Equal numbers (1 × 10 3 cells each) of congenically distinct P14 CD8 T cells, including each sorted subset and freshly isolated P14 CD8 T cells (control), were adoptively co-transferred into B6 mice, followed by LCMV-Arm infection. The responses of sorted and control P14 populations in the blood were monitored over time. (B) Kinetics of the frequency of sorted and control P14 CD8 T cells. The frequency of control P14 cells at day 8 post-infection was set as 100. Each symbol represents the mean, and error bars indicate the SEM. (C) Experimental design for panels D and E. IL18Rα Lo CD73 Lo (DN: double negative) or IL18Rα Hi CD73 Hi (DP: double positive) CD44 Lo naïve CD8 T cells were sorted from splenocytes of B6 mice (CD45.2 +/+ ). The number of DbGP33 tetramer + CD8 T cells was confirmed using a portion of the sorted cells via tetramer enrichment. Bulk sorted cells, containing equal numbers of DbGP33 tetramer + cells, were separately transferred into recipient mice (CD45.1 + CD45.2 + ), followed by LCMV-Arm infection. On days 7–8 post-infection, tetramer enrichment was performed to analyze the response of donor-derived DbGP33 + CD8 T cells in the spleen. (D) Representative flow plots. (E) Frequency of donor-derived DbGP33 + CD8 T cells among total DbGP33 + CD8 T cells in the spleen. Each symbol represents an individual mouse. Data were pooled from 2 independent experiments with 5 or 9 mice per group. Statistical analyses were performed using one-way ANOVA (B) and unpaired t test (E). * p < 0.05; ** p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Article Snippet: To prevent anti- CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti- CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques: Transgenic Assay, Isolation, Control, Infection, Derivative Assay

Journal: bioRxiv

Article Title: Functional heterogeneity and plasticity in naïve CD8 T cells drive superior effector and memory responses

doi: 10.1101/2025.10.16.682909

Figure Lengend Snippet: IL-18Rα Hi CD73 Hi CD44 Lo and IL-18Rα Lo CD73 Lo CD44 Lo CD8 T cells were sorted from splenocytes of naïve B6 mice.

Article Snippet: To prevent anti- CD44 antibody-mediated depletion of sorted cells after adoptive transfer, recombinant anti- CD44 antibody (clone: REA664, Miltenyi Biotec), that has a mutated Fc region, was used.

Techniques:

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: Identification of the target of sTN58 aptamer on TNBC cell surface. (A) Schematic representation of biotin-sTN58-mediated affinity purification. Membrane-protein fraction from Cis-Pt-R cells were subjected to a preclearing step to remove non-specific components prior to the sTN58-mediated precipitation. The colloidal Blue-stained SDS-PAGE (10 %) displayed is utilized for the analysis of target purification mediated by the sTN58 aptamer. The molecular weights of protein markers are reported. Lane 1, molecular markers; lane 2, membrane extracts; lane 3, 15 μg aliquot of unbound proteins from SCR-mediated purification; lane 4, proteins captured with sTN58. Red boxes indicate the regions excised for MS analyses. (B) Comparison of transcript expression values of best candidates in different BC cell lines. The normalized transcript expression values (nTPM), according to HPA, are reported relative to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. Box indicates the 5 candidates chosen for experimental validation. (C) Immunoblot analysis of EphA2, CD44, integrin β1, myoferlin, liprin β1 and ZO-1, and of the housekeeping proteins α-tubulin and vinculin. The molecular weights of protein markers are reported. Black dashed lines delineate the boundary between non-contiguous lanes of the same gel. (D) The histogram shows the relative fold-change in expression levels of the indicated proteins compared to the housekeeping protein α-tubulin or vinculin, normalized to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. (E) Binding affinity (1/Kd) of TN58 aptamer to the indicated cell lines expressed relative to MDA-MB-231 target cells. Dose response curves and binding affinity calculations for MDA-MB-231 and Cis-Pt-R and Dox-R chemoresistant cells, as well as non-TNBC BT-474, MCF-7 and A431 cells were previously reported [ , ]. The dose response curve used for Kd calculation in relation to BT-549 is shown in . "NB", no binding.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Affinity Purification, Membrane, Staining, SDS Page, Purification, Comparison, Expressing, Biomarker Discovery, Western Blot, Binding Assay

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: sTN58 binds to CD44-positive TNBC cell lines. Following 5 min incubation at RT with 2 μM Alexa 647-sTN58, Cis-Pt-R (A) or BT-474 (B) cells were stained with CD44 Ab, visualized by confocal microscopy, and photographed. Alexa 647-SCR was used as a negative control. Alexa 647-sTN58, CD44 Ab and nuclei are visualized in red, green, and blue, respectively. Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. Co-localization results appear yellow in the merged images (Overlap Coefficient 0.74). All digital images were captured under identical settings to allow direct comparison of staining patterns. (C) Flow cytometry analyses of Cis-Pt-R, MDA-MB-231, BT-549 and BT-474 cells treated with CD44-PE Ab or Alexa 647-sTN58. (D) Quantification of the geometric mean fluorescence intensity (gMFI) of Alexa 647-sTN58- or CD44-PE Ab-treated cells normalized to the gMFI of the untreated cells. Bars depict mean ± SD of at least two independent experiments. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Incubation, Staining, Confocal Microscopy, Negative Control, Comparison, Flow Cytometry, Fluorescence

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: CD44 silencing results in reduced sTN58 binding. (A, E) Cis-Pt-R (A) and BT-549 (E) cells were left untreated or transfected with si-CD44 or siRNA ctrl. At 48 h post-transfection, cells were harvested, and cell lysates prepared and immunoblotted with CD44 Ab. Equal loading was confirmed by immunoblot with anti-α-tubulin antibody. Molecular weights of protein markers are reported. (B, F) The histogram depicts the densitometric ratio of CD44 expression to α-tubulin. Values are shown relative to the untreated control, arbitrarily set to 1. ∗ P < 0.05, ∗∗ P < 0.01 relative to siRNA ctrl. (C, G) Binding of CD44-PE Ab ( left ) and Alexa 647-sTN58 ( right ) to Cis-Pt-R (C) and BT-549 (G) cells following 48 h transfection with si-CD44 (green) and siRNA ctrl (gray). (D, H) The histogram shows gMFI of si-CD44-transfected cells treated with sTN58 aptamer or CD44 Ab, normalized to the gMFI of untreated cells, and expressed as percentage with respect to siRNA ctrl-transfected cells. ∗∗∗∗ P < 0.0001 relative to siRNA ctrl. (I) Immunoblot analysis of CD44 and the housekeeping protein α-tubulin. The molecular weights of protein markers are reported. (J) The histogram shows the relative fold-change in CD44 expression levels compared to α-tubulin, normalized to MDA-MB-231 target cells, whose expression level is arbitrarily set to 1. ∗ P < 0.05, ∗∗∗ P < 0.001 relative to MDA-MB-231. (K) Flow cytometry analyses of Cis-Pt-R, MCF 10A, THP-1 and HS-5 cells treated with Alexa 647-sTN58. (L) Quantification of the gMFI of Alexa 647-sTN58-treated cells normalized to the gMFI of the untreated cells. ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant. In B, D, F, H, J, L, bars depict mean ± SD of at least two independent experiments.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Binding Assay, Transfection, Western Blot, Expressing, Control, Flow Cytometry

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: sTN58 aptamer and CD44 Ab colocalize with integrin β1 Ab on Cis-Pt-R cells. (A) Following 5 min incubation at RT with 2 μM Alexa 647-sTN58, Cis-Pt-R cells were stained with integrin β1 Ab, visualized by confocal microscopy, and photographed. (B) Cell lysates from Cis-Pt-R cells left untreated or treated for 48 h with 100 nM siRNA ctrl or si-ITGB1 were analyzed by immunoblotting with integrin β1, CD44 and anti-α-tubulin antibodies. Molecular weights of protein markers are reported. (C) The histogram shows the protein expression/α-tubulin ratio based on the densitometric signals. Values are shown relative to untreated samples, arbitrarily set to 1. (D) Binding of integrin β1-APC-Cy7 Ab ( left ) and Alexa 647-sTN58 ( right ) to Cis-Pt-R cells following 48 h transfection with siRNA ctrl (gray) and si-ITGB1 (pink). (E) The histogram shows gMFI of si-ITGB1-transfected cells treated with Alexa 647-sTN58 or integrin β1-APC-Cy7 Ab, normalized to the gMFI of untreated cells, and expressed as percentage with respect to siRNA ctrl-transfected cells. Bars depict mean ± SD of two independent experiments. ∗∗∗ P < 0.001; ns, no significant. (F) Confocal microscopy analyses of A431 cells treated with sTN58 and stained with integrin β1 Ab, as in A, or stained with CD44-PE and integrin β1 antibodies. Alexa 647-SCR was used as a negative control. In A, F, aptamers and CD44-PE Ab are visualized in red, integrin β1 Ab in green and nuclei in blue. All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. Co-localization results appear yellow in the merged images. Arrowheads indicate some co-localization points between sTN58 and integrin β1 Ab (Overlap Coefficient, 0.80). (G) Flow cytometry analyses of A431 cells treated with CD44-PE Ab, Alexa 647-sTN58 and integrin β1-APC-Cy7 Ab. (H) Quantification of the gMFI of sTN58-, CD44 PE- and integrin β1-APC-Cy7-treated cells normalized to the gMFI of the untreated cells. Bars depict mean ± SD of three independent experiments. ∗∗∗∗ P < 0.0001 relative to untreated cells; ns, no significant.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Incubation, Staining, Confocal Microscopy, Western Blot, Expressing, Binding Assay, Transfection, Negative Control, Comparison, Flow Cytometry

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: sTN58 inhibits invasive growth and vessel like-structures formation in 3D cell cultures. (A) Representative phase-contrast images of Cis-Pt-R cells grown in 2D or in Matrigel (3D) in the presence of 500 nM sTN58 or SCR for the indicated time points. (B) The invasive ability of Cis-Pt-R cells at 5 days is expressed as the number of colonies and branches per field. (C, D) Invasion of Cis-Pt-R (C) and Dox-R (D) cells toward 10 % FBS was analyzed by transwell invasion assay in the presence of 500 nM sTN58 or SCR for 72 h. Photographs of a representative experiment are shown. (E, F) Data are presented as percentage of invaded cells in the presence of sTN58 compared with SCR control. (G, I) Representative phase-contrast images of Cis-Pt-R cells (G) and Dox-R (I) grown on Matrigel in the presence of HA and treated with 500 nM sTN58 or SCR for 24 h. (H, J) Tube formation ability is expressed as the number of junctions and loops per field. (A, C, D, G, I) Magnification 10×, scale bar = 200 μm. (B, E, F, H, J) Bars depict means ± SEM of at least two independent experiments. ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 relative to SCR-treated cells. (K) AF3 molecular docking model of sTN58-CD44 binding complex; sTN58, HA-binding site and CD44 folded domain are shown in blue, white and green, respectively.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Transwell Invasion Assay, Control, Binding Assay

Journal: Bioactive Materials

Article Title: A novel CD44-targeting aptamer recognizes chemoresistant mesenchymal stem-like TNBC cells and inhibits tumor growth

doi: 10.1016/j.bioactmat.2025.04.027

Figure Lengend Snippet: sTN58 selectively targets CD44-positive 4T1 xenografts. (A) Representative confocal images of 4T1 (8.0 × 10 4 cells) incubated with 2 μM Alexa 647-sTN58 or Alexa 647-SCR and then fixed and stained with CD44 Ab. Aptamers, CD44 Ab and nuclei are visualized in red, green and blue, respectively. Co-localization results appear yellow in the merged images (Overlap Coefficient, 0.76). All digital images were captured at the same setting to allow direct comparison of staining patterns. Magnification 63× , 1.0× digital zoom, scale bar = 10 μm. (B) Binding of CD44-PE Ab ( left ) and Alexa 647-sTN58 ( right ) to 4T1 cells using flow cytometry. (C) Quantification of the gMFI of sTN58 aptamer- or CD44 Ab-treated cells normalized to the gMFI of the untreated cells. Bars depict mean ± SD of two independent experiments. ∗∗ P < 0.01, ∗∗∗ P < 0.001 relative to untreated cells. (D) Mice bearing mammary fat pad orthotopic 4T1 tumors were i.v. injected with 0.75 nmol of either NIR-sTN58 or NIR-SCR and analyzed with FMT at the indicated time points; Pre, before injection. Representative volume renderings taken at the same color gating for NIR-sTN58 and NIR-SCR injected mice are shown. (E) The amount of fluorescence (pmol) was quantified in specific VOIs encompassing the tumor in the animal. (F) Representative ex vivo FRI imaging of tumor and major organs (liver, kidneys, spleen, lung, heart and muscle) harvested from mice at 24 h post-injection of NIR-sTN58 and NIR-SCR. (G) The histogram indicates the mean FRI Signal Intensity of tumors and organs in the two groups. (E, G) Bars depict mean ± SD. ∗∗ P < 0.01; ∗∗∗ P < 0.001 relative to NIR-SCR; ns, no significant. (H) Plasma pharmacokinetic profile of sTN58. Concentration of aptamer is shown as a function of time following a single i.v. injection in Balb/c mice. Data are presented as the mean ± SEM.

Article Snippet: For cell binding of anti-CD44 antibody, 2.0 × 10 5 cells were left untreated or incubated for 20 min at RT with CD44 phycoerythrin (PE)-conjugated antibody (CD44-PE Ab, IM7, 0.25 μg/test, Santa Cruz Biotechnology) in DPBS supplemented with 0.1 % bovine serum albumin.

Techniques: Incubation, Staining, Comparison, Binding Assay, Flow Cytometry, Injection, Fluorescence, Ex Vivo, Imaging, Clinical Proteomics, Concentration Assay