Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 1. ROR1 mRNA expression in B-CLL. A, ROR1 mRNA versus ZAP-70 mRNA expression. A cohort of 107 B-CLL patient samples (79 IgVH mutated and 28 IgVH unmutated) was analyzed for ROR1 versus ZAP-70 mRNA expression by gene expression profiling. ROR1 values on the y axis are given as fold expression in log2 over the background expression level in nine pooled lymphoma cell lines (5). ZAP-70 values on the x axis are centered to the median of all samples. Dashed line, scaled cutoff between ZAP-70 ^ negative (left) and ZAP-70 ^ positive (right) samples as described (5).The median ROR1 mRNA expression was 6.5-fold higher (2.7 log2) in the B-CLL patient samples compared with the nine pooled lymphoma cell lines. No significant difference in median ROR1 mRNA expression was found between ZAP-70 ^ negative and ZAP-70 ^ positive (P = 0.906) or between IgVH-mutated and IgVH-unmutated (P = 0.176) B-CLL patient samples. B, detection of theT518M polymorphism of ROR1by RT-PCR followed by DNA sequencing. Left, predicted domain structure of ROR1protein (12). Numbers, amino acid sequence of ROR1protein before signal peptide cleavage. Right, chromatograms from three representative mRNA samples prepared from B-CLL PBMC and amplified by RT-PCR.Top, uniform ACG (threonine) allele expression found in 8 of 15 B-CLL patient samples; center, uniform ATG (methionine) allele expression found in 1of 15 B-CLL patient samples; bottom, ACG/ATG allele expression mixture found in 6 of15 B-CLL patient samples.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Sequencing, Amplification

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 2. ROR1protein expression on the surface of B-CLL cells. A, flow cytometry profiles of PBMC from a representative B-CLL patient. Four-color flow cytometry was carried out with a mouse anti-human CD19 mAb conjugated to PE, a mouse anti-human CD5 mAb conjugated toAPC, gahROR1pAb or gahROR2 pAb, swine anti-goat-FITC conjugate, and propidium iodide.The histograms on the right represent staining for ROR1 (green) and ROR2 (pink) in CD5+ CD19+ B-CLL cells (top) and CD5+ CD19- Tcells (bottom).The background signal with normal goat immunoglobulin or secondary antibody alone is shownin black. B, biotinylated cell surface proteins from B-CLL cells or normal B cells were separated by SDS-PAGE. Subsequent Western blotting with gahROR1pAb and donkey anti-goat-HRP conjugate detected a single band of f120 kDa in B-CLL cells (left lane). No band was detected in normal B cells (right lane). C, B-CLL cells were incubated with gahROR1pAb for1h at 4jC. Subsequently, the cells were washed and either left at 4jC (green) or incubated for 1h at 37jC in the absence (red) or presence (blue) of 3 Amol/L phenylarsine oxide, followed by flow cytometry analysis with swine anti-goat-FITC conjugate.The background signal with secondary antibody alone is shown in black.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Expressing, Flow Cytometry, Staining, SDS Page, Western Blot, Incubation

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 5. Normal adult tissues do not express cell surface ROR1. Lysates from PBMC of B-CLL patients (5 Ag total protein per lane) were compared with lysates from 28 normal adult tissues (20 Ag total protein per lane) byWestern blotting using gahROR1 pAb followed by donkey anti-goat-HRP conjugate. Arrows, f120-kDa band that specifies cell surface ROR1 (Fig. 2B). Rabbit anti-human glyceraldehyde-3- phosphate dehydrogenase pAb followed by donkey anti-rabbit-HRP conjugate were used as positive control (bottom). Normal adult tissues: Brn, brain; Brt, breast;Tes, testis; Ova, ovary; Ute, uterus; Pro, prostate; Lyn, lymph node; Int, small intestine; Hrt, heart; Liv, liver; Lun, lung; Kid, kidney; Pan, pancreas; Adi, adipose; Skn, skin; Spl, spleen; Adr, adrenal gland; Amy, amygdala; Pit, pituitary gland; Pla, placenta; Bla, bladder;Ton, tonsil; Sto, stomach; Col, colon; Rec, rectum; Mus, skeletal muscle; Spn, spinal cord; Eye, eye.

Article Snippet: Affinity-purified polyclonal goat anti-human ROR1 (gahROR1 pAb), polyclonal goat anti-human ROR2 antibodies (gahROR2 pAb), and polyclonal goat anti-human CD5 antibodies (gahCD5 pAb) were used as primary antibodies (R&D Systems).

Techniques: Positive Control

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 1. ROR1 mRNA expression in B-CLL. A, ROR1 mRNA versus ZAP-70 mRNA expression. A cohort of 107 B-CLL patient samples (79 IgVH mutated and 28 IgVH unmutated) was analyzed for ROR1 versus ZAP-70 mRNA expression by gene expression profiling. ROR1 values on the y axis are given as fold expression in log2 over the background expression level in nine pooled lymphoma cell lines (5). ZAP-70 values on the x axis are centered to the median of all samples. Dashed line, scaled cutoff between ZAP-70 ^ negative (left) and ZAP-70 ^ positive (right) samples as described (5).The median ROR1 mRNA expression was 6.5-fold higher (2.7 log2) in the B-CLL patient samples compared with the nine pooled lymphoma cell lines. No significant difference in median ROR1 mRNA expression was found between ZAP-70 ^ negative and ZAP-70 ^ positive (P = 0.906) or between IgVH-mutated and IgVH-unmutated (P = 0.176) B-CLL patient samples. B, detection of theT518M polymorphism of ROR1by RT-PCR followed by DNA sequencing. Left, predicted domain structure of ROR1protein (12). Numbers, amino acid sequence of ROR1protein before signal peptide cleavage. Right, chromatograms from three representative mRNA samples prepared from B-CLL PBMC and amplified by RT-PCR.Top, uniform ACG (threonine) allele expression found in 8 of 15 B-CLL patient samples; center, uniform ATG (methionine) allele expression found in 1of 15 B-CLL patient samples; bottom, ACG/ATG allele expression mixture found in 6 of15 B-CLL patient samples.

Article Snippet: A 96-well half area plate (Costar 3690) was coated with 25 AL/well of 2 Ag/mL rat antihuman ROR1 mAb (R&D Systems) overnight at 4jC.

Techniques: Expressing, Gene Expression, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing, Sequencing, Amplification

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 2. ROR1protein expression on the surface of B-CLL cells. A, flow cytometry profiles of PBMC from a representative B-CLL patient. Four-color flow cytometry was carried out with a mouse anti-human CD19 mAb conjugated to PE, a mouse anti-human CD5 mAb conjugated toAPC, gahROR1pAb or gahROR2 pAb, swine anti-goat-FITC conjugate, and propidium iodide.The histograms on the right represent staining for ROR1 (green) and ROR2 (pink) in CD5+ CD19+ B-CLL cells (top) and CD5+ CD19- Tcells (bottom).The background signal with normal goat immunoglobulin or secondary antibody alone is shownin black. B, biotinylated cell surface proteins from B-CLL cells or normal B cells were separated by SDS-PAGE. Subsequent Western blotting with gahROR1pAb and donkey anti-goat-HRP conjugate detected a single band of f120 kDa in B-CLL cells (left lane). No band was detected in normal B cells (right lane). C, B-CLL cells were incubated with gahROR1pAb for1h at 4jC. Subsequently, the cells were washed and either left at 4jC (green) or incubated for 1h at 37jC in the absence (red) or presence (blue) of 3 Amol/L phenylarsine oxide, followed by flow cytometry analysis with swine anti-goat-FITC conjugate.The background signal with secondary antibody alone is shown in black.

Article Snippet: A 96-well half area plate (Costar 3690) was coated with 25 AL/well of 2 Ag/mL rat antihuman ROR1 mAb (R&D Systems) overnight at 4jC.

Techniques: Expressing, Flow Cytometry, Staining, SDS Page, Western Blot, Incubation

Journal: Clinical Cancer Research

Article Title: Unique Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Human B-Cell Chronic Lymphocytic Leukemia

doi: 10.1158/1078-0432.ccr-07-1823

Figure Lengend Snippet: Fig. 5. Normal adult tissues do not express cell surface ROR1. Lysates from PBMC of B-CLL patients (5 Ag total protein per lane) were compared with lysates from 28 normal adult tissues (20 Ag total protein per lane) byWestern blotting using gahROR1 pAb followed by donkey anti-goat-HRP conjugate. Arrows, f120-kDa band that specifies cell surface ROR1 (Fig. 2B). Rabbit anti-human glyceraldehyde-3- phosphate dehydrogenase pAb followed by donkey anti-rabbit-HRP conjugate were used as positive control (bottom). Normal adult tissues: Brn, brain; Brt, breast;Tes, testis; Ova, ovary; Ute, uterus; Pro, prostate; Lyn, lymph node; Int, small intestine; Hrt, heart; Liv, liver; Lun, lung; Kid, kidney; Pan, pancreas; Adi, adipose; Skn, skin; Spl, spleen; Adr, adrenal gland; Amy, amygdala; Pit, pituitary gland; Pla, placenta; Bla, bladder;Ton, tonsil; Sto, stomach; Col, colon; Rec, rectum; Mus, skeletal muscle; Spn, spinal cord; Eye, eye.

Article Snippet: A 96-well half area plate (Costar 3690) was coated with 25 AL/well of 2 Ag/mL rat antihuman ROR1 mAb (R&D Systems) overnight at 4jC.

Techniques: Positive Control

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: (A) Flow cytometry analysis of ROR1 protein expression on the surface of NSCLC cell lines. The histograms on the right represent staining for ROR1 with polyclonal goat anti-ROR1 antibody. The background signal with normal goat IgG is shown in gray shadow. (B) Representative images of lung adenocarcinoma tissues (LA) detected by immunohistochemical analysis. Formalin-fixed, paraffin-embedded tissues were stained with polyclonal rabbit anti-ROR1 antibody or normal rabbit IgG. Tissue-bound ROR1 is shown in brown and the nucleus counterstained with hematoxylin is in blue (scale bar in the bottom right picture represents 50μm). A score of 0 indicates that none of the cells within the sample bound to the anti-ROR1 pAb; a score of 1 indicates low-level binding of the pAb on more than 25% of tumor cells; a score of 2 indicates low-level binding of the pAb on more than 50% of tumor cells or moderate-level staining on more than 25% of tumor cells; a score of 3 indicates moderate-level staining on more than 75% of tumor cells or high level staining on more than 50% of tumor cells. Controls include: Chronic lymphocytic leukemia lymph node (CLL LN) stained with normal rabbit IgG as negative control and with polyclonal rabbit anti-ROR1 antibody as positive control; Reactive hyperplasia of lymph node (RH LN) and tissues judged to be adjacent to cancer (TAC) stained with polyclonal rabbit anti-ROR1 antibody as negative controls. (C) A summary of immunohistochemical analysis for ROR1 staining in lung adenocarcinoma specimens. The proportion of lung tumor tissues found negative (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in the pie chart. (D-E) The proportion of lung adenocarcinoma tissues of different stages or sex found lacking staining (Score 0) or having weak (Score 1), moderate (Score 2) or strong staining (Score 3) for ROR1 is indicated in each bar. The number of different cases examined for each group is indicated in the parentheses. Statistical analyses was performed using Mann–Whitney U test. p = 0.048.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Flow Cytometry, Expressing, Staining, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Binding Assay, Negative Control, Positive Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: (A-C) PC9, XLA-07, and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for ROR1 protein expression with chimeric rabbit/human anti-ROR1 monoclonal antibody R12 by flow cytometry (A), growth-inhibition by MTS assay (B) and apoptosis induction by Annexin-V/PI staining (C). The height of each bar in the graph B provides the mean number of viable cells that are representative of more than three independent experiments. *** p<0.001, ** p<0.01 by Student’s t test.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Control, Expressing, Flow Cytometry, Inhibition, MTS Assay, Staining

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: PC9 and NCI-H1975 were treated with ROR1 siRNA (siROR1) or control siRNA (siControl) for 72 h and examined for phosphorylated AKT at Ser-473 (p-AKT), phosphorylated mTOR at Ser-2448, and phosphorylated PTEN at Ser-380/Thr-382/383 (p-PTEN) by immunoblot analysis.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Control, Western Blot

Journal: PLoS ONE

Article Title: Silencing of Receptor Tyrosine Kinase ROR1 Inhibits Tumor-Cell Proliferation via PI3K/AKT/mTOR Signaling Pathway in Lung Adenocarcinoma

doi: 10.1371/journal.pone.0127092

Figure Lengend Snippet: Activation of ROR1 significantly enhanced phosphorylation of both AKT and mTOR, and deactivated PTEN, a negative regulator of PI3K/AKT.

Article Snippet: Five μg/ml of polyclonal goat anti-ROR1 antibody (R&D system, Minneapolis, MN, USA) or chimeric rabbit/human anti-ROR1 monoclonal antibody R12 with HA tag which was developed by the correspondence author in Christoph Rader’s lab [ ], or normal goat IgG (Santa Cruz Biotechnology, Dallas, TX, USA) was added to the cells and incubated on ice for 30 min before washing twice with flow cytometry buffer.

Techniques: Activation Assay, Phospho-proteomics

Journal: Chemmedchem

Article Title: Novel Para ‐Phenylenediamine‐Based Derivatives as Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) Inhibitors: An In Vitro Preliminary Characterization

doi: 10.1002/cmdc.202500247

Figure Lengend Snippet: Known chemical structures of ROR1 inhibitors.

Article Snippet: Recombinant human ROR1 protein (Fc Tag) and recombinant human ROR1 protein (aa 453‐783, His&GST Tag) were purchased from Elabscience (Texas, USA).

Techniques:

Journal: Chemmedchem

Article Title: Novel Para ‐Phenylenediamine‐Based Derivatives as Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) Inhibitors: An In Vitro Preliminary Characterization

doi: 10.1002/cmdc.202500247

Figure Lengend Snippet: Structure‐based drug design approach leading to the para‐ phenylenediamine derivatives acting as ROR1 inhibitors.

Article Snippet: Recombinant human ROR1 protein (Fc Tag) and recombinant human ROR1 protein (aa 453‐783, His&GST Tag) were purchased from Elabscience (Texas, USA).

Techniques:

Journal: Chemmedchem

Article Title: Novel Para ‐Phenylenediamine‐Based Derivatives as Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) Inhibitors: An In Vitro Preliminary Characterization

doi: 10.1002/cmdc.202500247

Figure Lengend Snippet: CETSA identifies ROR1 protein in the binding with 17 in JeKo‐1 cells. A) CETSA western blots and B) corresponding quantitative analysis of band intensity. 17 group was treated with 17 (40 μM) for 2 h and then subjected to 5 min incubation at the indicated temperature (45–58 °C). Ctrl group was treated with vehicle (0.1% DMSO) only in the same conditions. ROR1 level at 25 °C was set at 100%. Densitometry‐based quantification of western blotting signals was calculated by first normalizing to α ‐tubulin levels in individual samples. Statistical analyses were performed comparing the compound 17 group to the Ctrl group across different temperature points. Results are shown as mean ± SD from three independent experiments. ** and *** denote respectively p < 0.01 and p < 0.001 versus Ctrl.

Article Snippet: Recombinant human ROR1 protein (Fc Tag) and recombinant human ROR1 protein (aa 453‐783, His&GST Tag) were purchased from Elabscience (Texas, USA).

Techniques: Binding Assay, Western Blot, Incubation

Journal: Chemmedchem

Article Title: Novel Para ‐Phenylenediamine‐Based Derivatives as Receptor Tyrosine Kinase‐like Orphan Receptor 1 (ROR1) Inhibitors: An In Vitro Preliminary Characterization

doi: 10.1002/cmdc.202500247

Figure Lengend Snippet: Molecular effects of 17 on ROR1 pathways in JeKo‐1 cells. For each western blotting analysis, three independent experiments were performed, and the SD is expressed by error bars. The relative fold change versus untreated cells, set as 1, is shown in the graph. *, **, and *** denote respectively p < 0.05, p < 0.01, and p < 0.001 versus Ctrl.

Article Snippet: Recombinant human ROR1 protein (Fc Tag) and recombinant human ROR1 protein (aa 453‐783, His&GST Tag) were purchased from Elabscience (Texas, USA).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

doi: 10.1101/2023.07.13.548856

Figure Lengend Snippet: a, Representative images on day 1 and 8 of the MDA-MB-231 aggregates or fibroblast spheroids after tumor-on-chip perfusion with either control T or CAR-T cells, showing different degrees of T cell infiltration within the tumor chamber. MDA-MB-231 tumor cells express GFP and are pseudo-colored in blue, fibroblasts—representing non-malignant tissue—were labeled with CellTracker TM CMFDA and pseudo-colored in cyan, whereas T and CAR-T cells were labeled in CellTracker TM Deep Red. Yellow-dashed line marks the region of interest of each tumor aggregate/fibroblasts spheroid. MvECs were present in all chips. Scalebars: 200 µm. b, Quantification of mean intensity values of the control T and CAR-T cells within each tumor aggregate’s/fibroblast spheroid’s region of interest at different time points after (CAR-)T cell perfusion. “CAR-T control” indicates the condition where CAR-T cells were perfused through chips containing ROR1 − fibroblasts spheroids; n = 7-21 MDA-MB-231 tumor aggregates/fibroblast spheroids from 3-4 chips. Data are depicted as mean with ± SEM. **p<0.01, ***p<0.001, ****p<0.0001 as assessed by Bonferroni’s multiple comparisons test. c, MDA-MB-231 tumor aggregate growth post CAR-T cell treatment in comparison to (control) T cell condition as measured by quantifying the difference of each MDA-MB-231 aggregate area at different time points compared to their initial area before (CAR-)T cell perfusion; n = 12-16 aggregates from 4 chips. Depicted are mean ± SEM. d, Quantification of mean GFP intensity fold change expressed on each MDA-MB-231 tumor aggregate on day 1 after the perfusion of either (control) T or CAR-T cells. Each dot represents one tumor aggregate and black line indicates the mean value; n = 12-14 aggregates from 4 chips. ****p<0.0001; two-tailed unpaired t test. e, The bar graph shows the quantification of control T or CAR-T cells per tumor chamber on day 1 after the tumor-on-chip was perfused at a concentration of 100,000 (CAR-)T cells/mL. Empty chip was used as control; n = 7-8 chambers from 3 chips. Data are depicted as mean with ± SEM. *p<0.05; Bonferroni’s multiple comparisons test. Scalebar: 200 µm. f, Representative images on day 1 after perfusing the CAR-T cells (red) at either 100,000 or 500,000 cells/mL concentration through the tumor-on-chip containing MDA-MB-231 aggregates (green). The focal plane was set on the aggregates for all the acquisitions. Scalebar: 200 µm. f, Flow cytometry histograms showing ROR1 expression (red) in the MDA-MB-231 cells and fibroblasts compared with the unstained control (cyan).

Article Snippet: For the analysis of the expression of extracellular markers, the following antibodies were used depending on the experiment: anti-human ROR1 APC (130-118-015; Miltenyi Biotec), anti-human CD31 FITC (130-110-806; Miltenyi Biotec), anti-human CD3 PE/Cyanine7 (317334; BioLegend), anti-human CD4 APC/Fire750 (357426; BioLegend), anti-human CD8a PerCP (300922; BioLegend), anti-human CD69 FITC (310904; BioLegend) and anti-human CD25 BV421 (302630; BioLegend).

Techniques: Control, Labeling, Comparison, Two Tailed Test, Concentration Assay, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Solid tumor-on-chip model for efficacy and safety assessment of CAR-T cell therapy

doi: 10.1101/2023.07.13.548856

Figure Lengend Snippet: a, Flow cytometry histogram plots showing ROR1 expression in the high-ROR1-expressing PDOs (red), low-ROR1-expressing PDOs (cyan) compared with the unstained control (orange). b, Representative images on day 8 of the ROR1-low and -high expressing PDOs after perfused with either (control) T or CAR-T cells. Fluorescently-labeled T cells are pseudo-colored in red. The focal plane was set on the PDOs for all the acquisitions. Scalebar: 500 µm. c, Quantification of mean intensity values of the control T (depicted as “T” in the graph) and CAR-T cells within the region of interest of each PDO on day 8. MDA-MB-231 aggregates were used as positive control; n = 14-20 PDOs from 3-4 chips. Data are depicted as mean with ± SEM. ns: not significant, *p<0.05, **p<0.01; Dunn’s multiple comparisons test. d, Representative images of the ROR1-high and ROR1-low expressing PDOs (cyan) before CAR-T cell (red) perfusion and on day 8 of the experiment. Yellow-dashed line marks the region of interest of each PDO. Scalebar: 200 µm. e, Quantification of the levels of cytokines IL-2, IL-6, IL-10, TNF-α, IFN-γ and granzyme B in the effluents of the chips after 20 h of (control) T or CAR-T cells perfusion through the tumor-on-chip containing either MDA-MB-231 aggregates (positive control for CAR-T cell treatment), ROR1-high PDOs or ROR1-low PDOs; n = 3-4 chips. Data are depicted as mean with ± SEM. Each red dot represents one chip. f, Cytokine release kinetics of the abovementioned cytokines from day 1 to 6 after CAR-T cells perfusion from day 0 to 1 through the tumor-on-chip containing either MDA-MB-231 aggregates (positive control for CAR-T cell treatment), ROR1-high PDOs or ROR1-low PDOs; n = 4 chips. Data are depicted as mean with ± SEM. Dashed line indicates the lower limit of detection. Data points from day 8 are excluded as the values are mostly below the detection limit.

Article Snippet: For the analysis of the expression of extracellular markers, the following antibodies were used depending on the experiment: anti-human ROR1 APC (130-118-015; Miltenyi Biotec), anti-human CD31 FITC (130-110-806; Miltenyi Biotec), anti-human CD3 PE/Cyanine7 (317334; BioLegend), anti-human CD4 APC/Fire750 (357426; BioLegend), anti-human CD8a PerCP (300922; BioLegend), anti-human CD69 FITC (310904; BioLegend) and anti-human CD25 BV421 (302630; BioLegend).

Techniques: Flow Cytometry, Expressing, Control, Labeling, Positive Control