Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A A concentration range of Alexa Fluor 647-conjugated CD3xHER2 bsAbs varying in affinity and epitope recognition site on either arm, were added on top of collagen gels containing BT474 (HER2+) tumoroids. Localization of bsAbs to the tumoroids was analyzed after 24 h by confocal microscopy. Images show maximum projections. Bar = 100 μm. B Localization of three different bsAbs combining a CD3 high affinity arm with the indicated HER2 arms in the BT474 tumoroid. White arrows indicate penetration of bsAbs into the tumoroids. Blue, Hoechst; red, Alexa Fluor 647-conjugated bsAb. Images were obtained from a single z-section through the center of the tumoroid. Bar = 50 μm. C Cartoon showing the location on the HER2 receptor of the epitopes recognized by the different HER2 arms and whether the interactions are high or low affinity.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Concentration Assay, Confocal Microscopy

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs with either non-targeting CD3 arm or non-targeting HER2 arm. Bottom panel shows tumoroids treated with 10 μg/mL Cisplatin triggering near complete tumoroid killing versus tumoroids grown in absence of T-cells and bsAbs (negative control; NC) or T-cells only. Bar = 100 μm. B Quantification of image data as shown in A . Data were normalized to cisplatin condition. Graphs show mean and SEM of 3 independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). C Western blot showing loss of HER2 upon CRISPR-Cas9 mediated knockout in BT474 cells. Tubulin serves as a loading control. D Representative maximum projection images at 6 days after exposure of collagen embedded WT, shCTR, or sgHER2 BT474 tumoroids to a mixture of T-cells and 1 μg/mL CD3 wt xHER2 Herceptin bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. bar = 100 μm. E Quantification of image data as shown in D . Data were normalized to cisplatin condition. Graph shows mean and SEM of 3 independent experiments, each performed in triplicate. P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; *** P < 0.001.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Western Blot, CRISPR, Knock-Out, Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing tumoroid (blue), T-cells (green) and loss of viability detected by PI (red) 6 days after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and bsAbs for one representative experiment of at least 4 biological replicates. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of the image data as shown in A . Tumoroids grown in absence of T-cells and bsAbs are shown as negative control (NC). Tumoroids treated with 10 μg/mL Cisplatin are shown as positive control triggering near complete tumoroid killing. Results are normalized to cisplatin condition. Graphs show mean and SEM of 5 (CD3 wt xHER2 variants) or 4 (CD3 Low xHER2 variants) independent experiments, each performed in triplicate (3 individual wells each containing 1 collagen embedded BT474 tumoroid). P -value calculated using two-way ANOVA followed by Bonferroni’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to NC.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Negative Control, Positive Control

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Maximum projection images showing T-cell recruitment to the tumoroid and tumor killing with 12 h intervals from day 2 to day 5 after exposure of collagen embedded BT474 tumoroids to a mixture of T-cells and 0.1 μg/mL of the indicated bsAbs. Blue, tumor nuclei; green, T-cells; red, PI. Bar = 100 μm. B Quantification of time-lapse image data with 1-h intervals as shown in A . Green line represents the number of T-cells localized in the tumoroid. Red line represents the percentage of PI positive tumor cells, indicating loss of viability. Mean and SEM of three tumoroids is shown.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques:

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Cartoon illustrating experimental setup: BT474 tumoroids were printed below the higher surface of collagen gels with a diagonal upper surface. A mixture of T-cells and bsAbs was added on top of the lower surface. Maximum projection images are shown for bsAbs with a CD3 high affinity arm in combination with a high affinity HER2 arm either recognizing epitope 169 or epitope 153. BsAbs were used at 1 μg/mL. Time lapse starts upon initial contact of T-cells with tumoroids (indicated by red circle; 36 h after adding T-cells and bsAbs). Confocal image stacks were captured every hour. A time span of 40 h is shown with an 8-h interval, followed by an image displaying the final time point (day 5) where T-cell mediated tumoroid killing is near complete. Blue, tumor nuclei; green, T-cells; Red, PI. B , C 3D T-cell tracking analysis of time-lapse image data as shown in A . B MSD analysis of two typical T-cells in the vicinity of the tumoroid. The MSD was determined for time-lag from 1 to 25 h. Red color indicates the presence of CD3 wt xHER2 169 and blue color indicates CD3 wt xHER2 153 . Insets depict the 3D track of the two T-cells. The MSD for the two cells were characterized by D B = 0.x ± 0.x um 2 /h, V B = 0.x ± 0.x um/h, and D R = 0.x ± 0.x um 2 /h, V R = 0.x ± 0.x um/h, respectively. C MSD analysis of the total population of T-cells in the vicinity of the tumoroid over increasing time-lag from 1 to 30 h. In total 25 and 35 trajectories were analyzed for CD3 wt xHER2 169 and CD3 wt xHER2 153 , respectively. Insets depict median and SD for the parameters diffusion (D), velocity (v), and diffusive fraction (f D ) for the population analysis for the indicated bsAbs. Note that f D is lower for CD3 wt xHER2 169 bsAb. P -value calculated using multi comparison Dunn’s test following non-parametric Kruskal–Wallis test. * P < 0.05. D Confocal images of a single z-section through the center of a tumoroid exposed to T-cells and either CD3 wt xHER2 169 or CD3 wt xHER2 153 bsAbs. Initial contact of T-cells with the tumoroid (36 h after adding T-cells and bsAbs) and the same area 8 h later is indicated by the white circle. Red arrow indicates T-cell infiltration occurring only with the CD3 wt xHER2 169 bsAb. Blue, tumor nucleus; red, PI; green, T-cell. Bar = 100 μm.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Cell Tracking Assay, Diffusion-based Assay, Comparison

Journal: Communications Biology

Article Title: CD3-engaging bispecific antibodies trigger a paracrine regulated wave of T-cell recruitment for effective tumor killing

doi: 10.1038/s42003-024-06682-9

Figure Lengend Snippet: A Schematic illustration of the transwell assay. BT474 cells were seeded in the lower chamber of a transwell plate, with or without the addition of unlabeled T-cells and with or without CD3 wt xHER2 169 bsAbs. Cell Tracker CMFDA-labeled T-cells were added to the upper chamber. The number of green-fluorescent T-cells migrating to the lower chamber was analyzed after 48 h using confocal microscopy and flow cytometry. A condition using 100 ng/mL CXCL12 was used as positive control. B Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) and green fluorescence channel images (showing labeled T-cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrow indicates a T-cell cluster. Bar = 100 μm. C Flow cytometry analysis of cell populations from the lower chamber at 48 h. Horizontal axis shows CMFDA signal (negative for unlabeled T-cells co-cultured with tumor cells in bottom chamber; positive for CMFDA-labeled T-cells recruited from the upper chamber); vertical axis shows CD3 levels. Flow cytometry analysis ( D ) and bar graph showing mean and SEM from 3 to 4 biological replicates ( E ), depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns, non-significant; ** P < 0.01; *** P < 0.001. F T-cell recruitment triggered by conditioned media (CM) from the indicated conditions. Graph shows cell counts of CMFDA-labeled infiltrated T-cells collected from the lower chamber. Mean and SEM from 2 to 4 biological replicates. P -value calculated using one - way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; * P < 0.05; ** P < 0.01. G Bright field images (showing a mixture of unlabeled T-cells, infiltrating CMFDA-labeled T-cells, as well as tumor cells) taken in the lower chamber at 48 h for the indicated conditions. Red dotted line outlines islands of tumor cells. Red arrows indicate T-cell clusters. Bar = 100 μm. H Bar graph showing mean and SEM from 3 biological replicates, depicting cell counts in Q2 (CMFDA:CD3 double positive T-cells migrated from the upper to the lower chamber) for the indicated conditions. P -value calculated using one-way ANOVA followed by Bonferroni’s multiple comparisons test. ns non-significant; ** P < 0.01.

Article Snippet: Human breast cancer cell lines BT474 and MDA-MB-231 were obtained from American Type Culture Collection (ATCC), authenticated using short tandem repeat (STR) profiling, and tested negative for mycoplasma prior to use.

Techniques: Transwell Assay, Labeling, Confocal Microscopy, Flow Cytometry, Positive Control, Fluorescence, Cell Culture