EMBO/soda-nel huggingface dataset:
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 2

Figure 2: Colorectal adenocarcinomas explants undergo collective invasion

A) Representative confocal images of non-invasive or invasive human colorectal cancer explants collected from 8 patients with NOS adenocarcinoma (EV2A and B). The explants were fixed 4 days after recovery and stained for the lumen (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii, iii and iv are displayed at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars show off-centered nuclei. Scale bars: 20 μm.

B, C) Graphs representing the percentage of adenocarcinoma explants displaying lumens (B) or protrusions (C) from 8 patients. Evaluation performed from DIC observation of live tissues, at least 50 explants were analysed per patients. The error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (ns: non significant).

D) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel monitored by DIC microscopy over 2 days (corresponding to Movie EV1). Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.

E) Images of an invading gland of a colorectal NOS adenocarcinoma, at the light microscopic level (i, semithin section, 100x objective), and as seen by electron microscopy (ii, 1000x magnification; and iii, 4000x magnification). Note that the bulk of the invading neoplastic gland is in a state of glandular organization (regions denoted by arrowheads in the left panel): epithelia (E, middle panel) are polarized to form a lumen (L), and are attached to a basal membrane (asterisk) by hemidesmosomes; the gland is surrounded by fibroblasts (F). However, this organization is focally disrupted at the tubular invasion pole (arrows in the left panel) where neoplastic epithelial cells have lost polarization and directly contact the surrounding stroma as the basal membrane is missing (right panel).
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 3

Figure 3: ROCK inhibition induces the collective invasion of Caco-2 cysts

A) Representative images of Caco-2 cysts transfected with control siRNA (siGlo) or siRNAs targeting ROCK1 and ROCK2, fixed and stained for prominin-1, F-actin (Phalloidin) and Nuclei (Hoechst). Confocal Z-sections of cysts are displayed. Arrowheads show non-protruding cells. Arrows point to protruding cells. White stars mark nuclei that are off-centered relative to the cyst's monolayer. Scale bars: 20 μm.

B) Representative confocal images of Caco-2 cysts treated with ROCK inhibitors Y27632 and H1152, non-treated (NT) or transduced with ROCK1 and ROCK2 shRNAs (shROCK1+2). The cysts were fixed 2 days after invasion and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i and ii are displayed at high magnification. White arrowheads point to non-protruding cells, green arrowheads point to the apical pole, arrows point to protruding cells and white stars show off-centered nuclei. Scale bar: 20 μm.

C) Bar graph representing the percentage of protrusive Caco-2 cysts from three independent experiments. Caco-2 cysts non-treated (NT), incubated with ROCK inhibitors Y27632 and H1152 or infected with lentiviruses encoding shRNA control and targeting ROCK1 and ROCK2. The cysts were fixed 2 days after invasion, over 100 cysts were counted for each condition. Error bar represents the Standard Error of the Mean (Means ± SEM). P values were calculated using unpaired t-test (***p<0.001, **p<0.01).

D) (Left panel) Schematic representation of the 3D Boyden chamber invasion assay. The Caco-2 were grown as cysts in 3D matrigel on the top chamber of the Boyden for 3 days, then the serum gradient was induced. Two days after, the nuclei of the cells that have reached the bottom side of the membrane were stained with Hoechst and quantified by automated acquisition and segmentation. (Right panel) DIC image of the membrane facing the top chamber reveals the cysts and the immunofluorescence image of the membrane facing the bottom chamber stained with Hoechst reveals the cells that have invaded.

E) The number of cells that have invaded from the top to the bottom chamber of the Boyden chamber was quantified by automated segmentation in non-treated (NT), in ROCK inhibited conditions (Y27632, H1152) or in cells transfected with siRNA against ROCK1 and ROCK2 (siROCK1+2). The number of cells that have invaded was normalized to control conditions and reported as the average invasion rate from at least three independent experiments (Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001, **p<0.01).

F) High magnification of boxed region from (D). The top (i) and the bottom (ii) side of the Boyden chamber membrane were stained for F-actin, E-cadherin, ZO-1, Ezrin and Hoechst. Arrow points to the protrusive cells of an invasive cyst and the white stars show the nuclei that engage in the protrusion. Scale bars: 40 μm.
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 4

Figure 4: ROCK inhibition promotes the collective invasion of colorectal adenocarcinoma explants

A) Representative images of organoids formed from patient derived xenografts (PDXs) that have been embedded in collagen-I gels during 3 days, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152). At end-point, organoids were stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). i and ii are high magnification of boxed regions of +Y27632 and +H1152 organoids respectively. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm.

B) The bar graph represents the percentage of protrusive organoids resulting from three independent experiments. Over 50 organoids were counted per condition (Means ± SEM). P values were calculated using paired t-test (***p<0.001).

C) Representative images of human adenocarcinoma explants after 2 days in 3D collagen-I gel, in non-treated (NT) or ROCK inhibitors treated-conditions (+Y27632) and (+H1152) and stained for the apical pole (Ezrin), F-actin (Phalloidin) and nuclei (DAPI). Boxed regions of NT, +Y27632, and +H1152 explants are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm.

D,E) Explants displaying (D) lumens or (E) protrusive cells from 10 patients were quantified and results presented as percentage of total explants. Over 50 explants were counted per condition (duplicate) and per patient(Means ± SEM). P values were calculated using unpaired t-test (****p<0.0001 , n.s : non significant).

F) Time-lapse sequences of an adenocarcinoma explant undergoing collective invasion into collagen-I gel in the presence of Y27632 monitored by DIC microscopy for 2 days (corresponding to Movie EV3). Arrowheads point to non-protruding cells, arrows point to protruding cells and white stars point to lumens. Scale bars: 50 μm.
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 5

Figure 5: ROCK2 inhibition is necessary and sufficient to control collective invasion

A) Caco-2 cysts expressing an empty vector (pLKO) or shROCK1 (#02) or shROCK2 (#36) after 2 days of invasion were fixed and stained for E-cadherin, F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnifications. Arrowheads point to non-protruding cells. Arrow points to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm.

B) The number of protrusive Caco-2 cyst was quantified in each of the mentioned conditions and represented in a bar graph as percentage of total cysts (Means ± SEM of at least 3 independent experiments, paired t-test, ***p<0.001, *p<0.05 n.s: non significant).

C) Caco-2 cysts stably expressing GFP alone or GFP-ROCK2DN#2, were submitted to a 2 days period invasion assay, then fixed and stained for F-actin (Phalloidin) and nuclei (DAPI). Boxed regions i, ii and iii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. White and black stars show nuclei that engage in protrusions. Scale bars: 20μm.

D) Bargraph representing the percentage of protrusive cysts transduced with GFP (CTRL, with or without Y27632) or with ROCK2DN#2 (Means ± SEM of at least 3 independent experiments, paired t-test, **p<0.01 n.s: non significant).

E) T84 cells grown in 3D organotypic culture form polarized cysts with a central lumen that display protruding leader cells at the basal pole (arrows) reminiscent of collective invasion. T84 cysts were transduced with lentiviruses encoding inducible GFP or Venus-ROCK2-DA and treated with doxycycline 1μg/ml. Scale bars: 20μm.

F) Bar graph representing the percentage of protrusive T84 cysts in control (WT), treated with ROCK inhibitor (Y27632) or transduced with lentiviruses expressing GFP or GFP-ROCK2-DA after induction by Doxycycline (Means ± SEM of at least 3 independent experiments) (paired t-test, ***p<0.001, *p<0.05).

G) Overall Survival Prognostic value of ROCK2 expression in colon cancer patients of the GSE33582 data set (n=477 patients with survival annotation) using Kaplan-Meier survival analysis. The patient cohort was divided into tumours with or without a down-regulation of ROCK2 relatively to non-tumoural tissues (expression cut-off value corresponds to the 1st decile of ROCK2 expression in non-tumoural sample, i.e. 7.6). Log-rank test p-values for survival distribution difference are indicated at 3 and 5 years.
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 6

Figure 6: ROCK2 inhibition promotes leader cell formation.

A) Schematic representation of Caco-2 cysts phenotypes obtain after mosaic transduction of ROCK2DN#2.

B) DIC acquisition of fixed mosaic Caco-2 cysts expressing GFP alone, GFP-ROCK2DN#1 or GFP-ROCK2DN#2. Boxed regions i and ii are shown at high magnification. Arrowheads point to non-protruding cells. Arrows point to protruding cells. Scale bars: 20μm.

C) Bar graph representing the percentage of mosaic Caco-2 cysts transduced with GFP or GFP-ROCK2DN#, with or without Y27632, harbouring a protruding morphology (Means ± SEM of at least 3 independent experiments, unpaired t-test, ***p<0.001, *p<0.05).

D) Quantification of the fate of the GFP positive cells, leader or follower, in mosaic cysts transduced with GFP-alone and treated with Y27632 (GFP+Y27632) or GFP-ROCK2DN#2 from 3 independent experiments (Means ± SEM, unpaired t-test, ***p<0.001).

E) Representative pull-down of RAC1-GTP by PAK-Crib, in Caco-2 cells treated with ROCK inhibitor Y27632, H1152 or blebbistatin and lysed at 12 or 16 hours. Immunoblot analyses were performed with anti-Rac1 and anti-GAPDH antibody from three independent experiments.

F) DIC acquisition of fixed Caco-2 cysts treated with ROCK inhibitors alone (Y27632 or H1152), or in combination with RAC1 inhibitor NSC23766. Arrowheads point to non-protruding cells and arrow point to protruding cell. Scale bar 20 μm.

G) Bar graph representing the percentage of protruding Caco-2 cysts in control conditions (non-treated, NT) or treated with NSC23766, Y27632 or H1152 (Means ± SEM of at least 3 independent experiments, unpaired t-test, ****p<0.0001, ***p<0.001, *p<0.05).
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 7

Figure 7: Caco-2 cysts collective invasion relies on the Rac-GEF FARP2.

A) siRNA screen performed in Caco-2 cysts treated with Y27632: Caco-2 cells transfected with control siRNA or pairs of siRNA targeting RhoGEFs were seeded in the modified 3D Boyden Chamber (Fig 3D) and grown as cysts for 3 days. Then, Caco-2 cysts were treated with Y27632 and the serum gradient was induced. After 2 days, the number of cells present on the bottom chamber were quantified by automated segmentation after Hoechst staining. The bar graph represents the mean invasion rate. Each condition was duplicated. The red dashed line represent the median invasion rate, the red full line indicates the 3 fold decrease in invasion rate. Three hits were identified: #2:ARHGEF18+RNEF, #7: SGEF+ARHGEF16 and #24: FARP1+FARP2 (Mean ±SD) .

B) Caco-2 cysts stably expressing empty vector (pGIPZ) or 2 distinct shFARP1 or shFARP2 were treated with Y27632, allowed to invade for 2 days, fixed and stained for F-actin (Phalloidin). Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells, arrows point to protruding cells. White stars show nuclei that engage in protrusions. Scale bars: 20μm.

C) Bar graphs representing the percentage of protrusive cysts formed from Caco-2 cells transduced with lentiviruses encoding pGIPZ or shRNA against FARP1 and FARP2 after treatment with Y27632 from at least 3 independent experiments (Means ± SEM, paired t-test, ns: non significant, **p<0.01).

D) Representative acquisition of Caco-2 cells overexpressing GFP-FARP2 grown in 2D and stained with ZO-1, in non-treated (NT) and after 2h with Y27632 or H1152. The dashed lines crossing the cell-cell junctions (i, ii and iii) are representative of the linescans measurements. Scale bars: 20μm.

E) Representative fluorescence intensity of ZO-1 (red) staining and GFP-FARP2 (green) along the dashed line i, ii and iii represented in Fig 7G corresponding to non-treated, Y27632 or H1152 treatments)

F) Bar graph representing the average ratio of GFP-FARP2 over ZO-1 fluorescence intensities measured with linescans crossing cell-cell junctions. The quantification was performed on 71 cells expressing GFP-FARP2-FL from 3 independent experiments. For each cell, between 10 and 15 lines (12 pixels wide) were drawn across random region of cell-cell junctions positive for ZO-1 staining (Mean ±SEM Unpaired t-test **p<0.01, *p<0.05).
ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinoma (2019)
Figure 8

Figure 8: FARP2 activation and Myosin-II inhibition cooperate to induce collective invasion downstream of ROCK2 inhibition.

A) Caco-2 cysts stably expressing GFP (treated or not with Y27632), or Cherry-FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH 730/733 (FARP2-del/Mut) were allowed to invade for 2 days, fixed and stained for F-actin. Boxed regions i, ii, iii, iv and v are shown at high magnification. Arrowheads point to non-protruding cells, arrow points to protruding cells, white star shows nucleus that engages in protrusion. Scale bars: 20μm.

B) Bargraph representing the percentage of protrusive cysts expressing GFP (treated or not with Y27632) or the mentioned GFP-FARP2 constructs from 3 independent experiments (Means ± SEM, paired t-test, **p<0,01, n.s: non significant).

C) Representative images of Caco-2 cysts treated with Y27632 or Blebbistatin or transfected with siRNA targeting Myosin-IIA and allowed to invade for 2 days. After fixation, the cysts were stained for F-actin (Phalloidin) and nuclei (DAPI). Representative Y27632-induced « invasive » phenotype and Blebbistatin-induced « dendritic » phenotype are displayed. Arrows point to protruding cells, white star shows nucleus that engages in protrusion and arrowheads point to "dendritic" protrusions.

D) Bar graph representing the percentage of cysts with the "invasive" or "dendritic" phenotypes after treatment with Y27632, Blebbistatin or siMyosin-IIA or siMyosin-IIB from at least 3 independent experiments (Means ± SEM, unpaired t-test,*p<0,05, n.s: non significant).

E) Mature Caco-2 cysts were transduced with GFP or Cherry tagged FARP2-full length (FARP2-FL), FARP2-730/733 mutant (FARP2-Mut) or FARP2 del PH/730/733 (FARP2-del/Mut), and treated (or not, NT) with Blebbistatin. 2 days after invasion,the cysts were fixed and stained for F-actin. Boxed regions i, ii, iii and iv are shown at high magnification. Arrowheads point to non-protruding cells; arrow points to protruding cells and white stars show nucleus that engage in protrusions. Scale bars: 20 μm.

F) Bargraph representing the percentage of protrusive Caco2 cysts expressing the mentioned constructs , treated or not with blebbistatin, from at least 3 independent experiments (Means ± SEM , t-test,**p<0,01, n.s: non significant).

SUPPLEMENTARY TABLE AND FIGURE LEGENDS

Supplementary Table 1: Effector library: Gene's names, accession numbers, siRNA catalog number and transfected pairs

Supplementary Table 2: Guanine nucleotide Exchange Factor library: Gene's names, accession numbers, siRNA catalog number, transfected pairs and raw data used for the graph in Fig 7D)
Identification of TAPBPL as a novel negative regulator of T cell function (2021)
Figure 2

Figure 2. The expression patterns of TAPBPL protein. (A, B) Analysis of TAPBPL protein expression on immune cells. Splenocytes from C57BL/6 mice were freshly harvested and analyzed for TAPBPL protein expression on resting immune cells. To obtain activated T cells, the splenocytes were incubated with anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies for 3 days. To obtain activated B cells, DCs, monocytes and macrophages, the splenocytes were incubated with LPS (10 µg/ml) for 3 days. The resting and activated immune cells were stained with anti-TAPBPL or isotype antibody (Ab), as well as anti-CD4, CD8, CD11b, F4/80, CD11c, CD19 or B220 antibody to identify immune cells. (A) Representative flow cytometric profiles and (B) statistical analysis showing the expression levels of TAPBPL protein on resting and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with isotype Ab; ** P<0.05 compared with resting cells. (C) Determination of TAPBPL protein expression in normal and tumor human tissues by immunohistochemistry using anti-TAPBPL or isotype Ab. (D) Statistical analysis of the expression levels of TAPBPL on tumor tissues. The data were presented as relative expression levels of TAPBPL on tumor tissues versus relative normal tissues (each group was normalized with isotype Ab first) (n=5). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with normal tissue. (E, F) Analysis of TAPBPL protein expression on the indicated cancer cell lines. (E) Representative flow cytometric profiles and (F) statistical analysis showing the expression levels of TAPBPL protein on the cancer cells (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with isotype Ab.
Identification of TAPBPL as a novel negative regulator of T cell function (2021)
Figure 3

Figure 3. The expression pattern of the putative TAPBPL receptor. (A, B) Splenocytes from C57BL/6 mice were freshly harvested. Resting and activated T cells, monocytes, macrophages, DCs and B cells were obtained as in Figure 2. The resting and activated immune cells were stained with biotinylated TAPBPL-Ig or control Ig，followed by streptavidin PE, as well as anti-CD4, CD8, CD11b, F4/80, CD11c, B220 or CD19 antibody to identify immune cells. (A) Representative flow cytometric profiles and (B) statistical analysis showing the binding TAPBPL-Ig or control Ig to freshly harvest and activated immune cells (n=3). Significance was calculated by two‐way ANOVA with Tukey test. * P<0.05 compared with control Ig. ** P<0.05 compared with resting cells. (C) HEK-293 cells were transfected with an expression vector containing the murine PD-1, CD28, BTLA, CTLA-4, or ICOS gene and screened for cells stably expressing each gene. The transfected cells were stained with antibodies against the respective PD-1, CD28, BTLA, CTLA-4, or ICOS protein (open histograms) or isotype Ab (shaded histograms). Representative flow cytometric profiles showing the expression of each receptor. (D) PD-1, CD28, BTLA, CTLA-4, or ICOS gene-transfected HEK-293 cells were stained with biotinylated TAPBPL-Ig and control Ig, followed by streptavidin-PE. Representative flow cytometric showing the binding of TAPBPL-Ig (open histograms) or control Ig protein (shaded histogram) to the transfected cells. The data are representative of 3 independent experiments.
Identification of TAPBPL as a novel negative regulator of T cell function (2021)
Figure 4

Figure 4. The effect of hTAPBPL-Ig protein on T cell activation in vitro. Splenic cells from C57BL/6 mice were cultured with (A, B, E, F, I, J) anti-CD3 antibody (1 µg/ml) or (C, D, G, H, K, L) anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig (10 and 15 µg/ml) or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for (A-D) 1 day or (E-L) 3 days. The cells were analyzed for (A-D) CD69+, (E-I, K) CD44hiCD62Llo cells, and (E, G, I-L) CD44loCD62Lhi cells in CD4 and CD8 T cells. (A, C, E, G, I, K) Representative flow cytometric and (B, D, F, H, J, L) statistical analyses of the (B, D) percentages of CD69+, and the numbers (X105) of (F, H, J, L) of CD44hiCD62Llo effector memory T cells and CD44loCD62Lhi Naïve T cells in CD4 and CD8 T cells. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.
Identification of TAPBPL as a novel negative regulator of T cell function (2021)
Figure 5

Figure 5. The effect of hTAPBPL-Ig protein on T cell proliferation in vitro. (A, B) T cells were purified from splenocytes of C57BL/6 mice by magnetic separation. The cells were cultured on plates pre-coated with (A) anti-CD3 antibody (1 µg/ml) or (B) anti-CD3 (1 µg/ml) and anti-CD28 (0.5 µg/ml) antibodies in the presence of graded doses of hTAPBPL-Ig (10 and 15 µg/ml) or equimolar amounts of control Ig (3.75 and 5.63 µg/ml) for 3 days. [3H] thymidine (1 µCi/well) was added to the cultures 12 hours before harvest. T cell proliferation was measured by [3H] thymidine incorporation. (C-E) Splenic cells were labelled with CFSE and cultured in 96-well plates that were precoated with anti-CD3 antibody and hTAPBPL-Ig or control Ig for 3 days as in (A). The cells were analyzed for CFSE levels by CD4+ and CD8+ T cells. (C) Representative flow cytometric analysis of CFSE distribution of CD4+ and CD8+ T cells, and (D, E) statistical analysis of (D) CD4 and (E) CD8 T cell proliferation. (F) Purified human T cells were cultured with plate-bound anti-human CD3 antibody (1 µg/ml) in the presence of graded doses of hTAPBPL-Ig (1.5 and 3 µg/ml) or control Ig protein (1.5 and 3 µg/ml) for 3 days. Cell proliferation was measured by [3H] thymidine incorporation. The data are expressed as mean + SD (n=3). Significance was calculated by two‐tailed Student's t‐test. * P<0.05 compared with control Ig.