itga6 isoforms  (Addgene inc)

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Graphs reporting the percentage of cell adhesion on different extracellular matrices of TOV-112D and OVSAHO PT-sen and PT-res pools measured by the quantitative cell adhesion CAFCA assay ( n = 2 in sextuplicate). Coll IV = Type IV Collagen; Coll I = Type I Collagen; FN = Fibronectin; LNs = Laminins; VN= Vitronectin. ( B ) Tables reporting the percentage of cells positive for the indicated integrins as measured by flow cytometry (FACS) in PT-sen and PT-res pools of TOV-112D and OVSAHO cells (ITGA1 = Integrin α1, ITGA2 = Integrin α2, ITGA5 = Integrin α5, ITGA6 = Integrin α6, ITGAV = Integrin αV, ITGB1 = Integrin β1, ITGB3 = Integrin β3, ITGB4 = Integrin β4). ( C ) FACS analyses of the expression profile of ITGB1, ITGB4, and ITGA6 integrins in OVSAHO PT-sen and PT-res clones. ( D ) Graphs reporting the percentage of cell adhesion on different extracellular matrices of the indicated TOV-112D and OVSAHO PT-sen and PT-res clones. ( n = 2 in sextuplicate). ( E ) Western blot analysis evaluating the expression of ITGA6 protein in tumor samples from the same patients taken at diagnosis (Pre) or at recurrence after chemotherapy (Post). Tubulin was used as loading control. ( F ) qRT-PCR analysis of ITGA6 mRNA expression in different EOC PT-sen and PT-res cells. ( G ) Western blot analysis of ITGA6 expression in TOV-112D PT-sen and PT-res clone exposed to a time-course treatment with CHX for the indicated times. Vinculin was used as loading control. ( H ) Graph reporting the expression of ITGA6 in OVSAHO PT-sen and PT-res cells treated or not with CDDP for 2 h. mRNA was analyzed by qRT-PCR at the indicated time points after CDDP removal. ( I ) Graphs reporting qRT-PCR analysis of mRNA expression of ITGA6 in TOV-112D ITGA6 HIGH and ITGA6 LOW subpopulations treated or not with CDDP. In ( F ), ( H ), and ( I ), mRNA levels were analyzed in triplicate and normalized to actin housekeeping gene and expressed in arbitrary units. In all the graph of the figure statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation.

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Flow Cytometry, Expressing, Clone Assay, Western Blot, Biomarker Discovery, Control, Quantitative RT-PCR, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Expression profile of β1 (ITGB1), β4 (ITGB4), and α6 (ITGA6) integrins in TOV-112D PT-sen and PT-res clones analyzed by flow cytometry (FACS). ( B ) Western blot analysis of ITGA6 expression in the indicated parental and PT-res EOC cells. GAPDH was used as loading control. ( C ) Graph (left) and representative western blot (right) reporting the expression of ITGA6 protein in total protein extracts of primary cells isolated from EOC patients’ ascites collected pre-treatment (primary) or after recurrence ( n = 34 from 27 patients). Vinculin was used as loading control. ( D ) Graph reporting TOV-112D parental cells sorted by FACS for ITGA6 HIGH (green) and ITGA6 LOW (purple) subpopulations. ( E ) Non-linear regression analyses of cell viability assay of TOV-112D PT-res, ITGA6 HIGH , and ITGA6 LOW subpopulations (described in D ) treated with increasing doses of CDDP for 72 h. Data are expressed as percentage of viable cells with respect to the untreated cells and represent the mean (± SD) of five replicates. The tables below show the IC50 and the Confidence Interval (CI) of each cell type. Fisher’s exact test was used to calculate the global p value reported in the graph. ( F ) Graphs reporting normalized expression mRNA of ITGA6 in ITGA6 HIGH and ITGA6 LOW subpopulations evaluated by qRT-PCR. ( G ) Graph reporting the normalized expression of ITGA6 in TOV-112D PT-sen and PT-res cells treated or not with CDDP for 2 h. mRNA was analyzed by qRT-PCR at the indicated time points after CDDP removal. In ( F ) and ( G ), mRNA levels were analyzed in triplicate and normalized to actin expression. In ( C ), ( F ), and ( G ), statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation. .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Expressing, Clone Assay, Flow Cytometry, Western Blot, Control, Isolation, Viability Assay, Quantitative RT-PCR, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Schematic representation of ITGA6 Full Length (FL) promoter cloned into pGL3-Luc vector. The c-Myc (blu) and SP1 (yellow) binding sites are indicated. The curved arrow indicates the translation initiation site. ( B ) Graph reporting the normalized luciferase activity measured in TOV-112D PT-sen cells transfected with ITGA6 FL promoter and treated or not (UNT) with CDDP for 24 h. ( C ) Graphs reporting the normalized expression of ITGA6 in TOV-112D PT-sen (left) and PT-res (right) cells treated with the SP1 inhibitor (Mithramycin, MTA) analyzed by qRT-PCR at the indicated time points. ( D ) Western blot analysis of ITGA6 expression in TOV-112D PT-sen cells treated with the SP1 inhibitor, MTA for the indicated time points. GAPDH was used as loading control. ( E ) Graphs reporting the normalized expression of ITGA6 mRNA in TOV-112D PT-sen (left) and PT-res (right) cells treated with the c-Myc inhibitor (10058-F4), analyzed by qRT-PCR at the indicated time points. ( F ) Graph reporting the luciferase activity of ITGA6 FL promoter measured in TOV-112D PT-sen treated with c-Myc inhibitor (10058-F4) or Sp1 inhibitor (MTA) for 24 h. In ( B ) and ( F ), data are expressed as fold value (on untreated in ( B ) and on DMSO in ( F ) of normalized luciferase activity and represent the mean (± SD) of three independent experiments. ( G ) Chromatin immunoprecipitation (ChIP) assay, using either anti-human c-Myc-or anti Sp1-specific antibodies, performed on TOV-112D PT-sen and PT-res cells treated or not with CDDP. Data are expressed as folds enrichment over the IgG, used as negative control and represent the mean of 5 replicates. ( H ) Western blot analysis of SP1-bound proteins in the chromatin fraction of TOV-112D PT-sen cells in the ChIP assay performed as in ( G ). ( I ) Pearson’s correlation analysis between ITGA6 and Sp1 protein expression in EOC tumor samples collected in our Institute (see Methods). ( J ) Sperman’s correlation analysis between ITGA6 and Sp1 mRNA expression in TCGA ovarian cancer dataset ( n = 489 samples) using the GEPIA on line tool. ( K ) Graph reporting the normalized ITGA6 mRNA expression in TOV-112D PT-sen cells treated with HDACs inhibitors for 24 h. (MS-275 inhibits preferentially HDAC1; CAY10683 inhibits preferentially HDAC2 and HDAC6; PCI-34051 inhibits preferentially HDAC8, M344 inhibits preferentially HDAC1 and HDAC6; Apicidin inhibits preferentially HDAC1 and HDAC3). ( L ) Graph reporting the ITGA6 mRNA expression in TOV-112D ITGA6 HIGH and ITGA6 LOW subpopulations treated with pan-HDAC inhibitor Panobinostat (Pano) or with the HDAC1 inhibitor MS-275 for 24 h. ( M ) Schematic representation depicting the proposed role of c-Myc/HDAC1/SP1 complex in the regulation of ITGA6 transcription. Under CDDP treatment the repressive c-Myc + HDAC1 complex is displaced leaving SP1 able to activate ITGA6 promoter, inducing its transcription. C-Myc and/or HDAC1 are constitutively displaced from ITGA6 promoter in PT-res cells. In ( C ), ( E ), ( K ), and ( L ), mRNA expression was analyzed in triplicate and normalized to actin. In ( B ), ( C ), ( E ), ( F ), ( G ), ( K ), and ( L ), statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation. .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Clone Assay, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Chromatin Immunoprecipitation, Negative Control, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A , B ) Graphs reporting the luciferase activity measured in TOV-112D PT-sen transfected with ITGA6 promoter full length (FL) together with SP1 ( A ) or c-Myc (MYC) ( B ) vectors. In A and B data are the mean (±SD) of three independent experiments. ( C ) Graphs reporting the expression of ITGA6 in OVSAHO PT-sen (left) and PT-res (right) cells treated with SP1 inhibitor (Mithramycin, MTA). mRNA expression was analyzed by qRT-PCR at the indicated time points. ( D ) Western blot analysis of ITGA6 expression in OVSAHO PT-sen cells treated with SP1 inhibitor, MTA for the indicated time points. ( E ) Western blot analysis of ITGA6 expression in TOV-112D PT-sen cells transfected with SP1 vector. Whole lysates were collected at the indicated time points after transfection. ( F ) Graphs reporting the expression of ITGA6 in OVSAHO PT-sen (left) and PT-res (right) cells treated with the c-Myc inhibitor (10058-F4)). mRNA was analyzed by qRT-PCR at the indicated time points. ( G ) ITGA6 promoter deletion mutants lacking c-Myc (Mut-1) or both c-Myc and SP1 binding sites (Mut-2) generated for this study. ( H ) Graph reporting the luciferase activity measured in TOV-112D PT-sen co-transfected with the full length (F.L.) or deletion mutants and the empty (black bars) or c-Myc (MYC, white bars) expression vectors. Data are the mean (±SD) of three independent experiments. ( I ) Western blot analysis of ITGA6 and SP1 protein expression in EOC tumor samples collected in our Institute (see Appendix Table S ). ( J ) Spearman’s correlation analysis between ITGA6 and c-Myc mRNA expression in TCGA ovarian cancer dataset ( n = 489 samples) using the GEPIA online tool. ( K ) Western blot analysis of c-Myc and SP1 protein expression in TOV-112D and OVSAHO PT-sen and PT-res clones. ( L ) Western blot analysis of ITGA6 protein expression in TOV-112D PT-sen and PT-res cells treated with a panel of epigenetic modifiers inhibitors for 24 h (UNC0631 and SGI-1027 = methyl transferase inhibitors; Panobinostat = pan-HDAC inhibitor; JQ1, iBET151 and Bromosporine = BET inhibitors; Methylstat = de-methyltransferase inhibitor; Tenovin= SIRTs inhibitor; Anacardic acid = HATs inhibitor). ( M ) Graph reporting the mRNA expression of ITGA6 in indicated PT-sen EOC cells treated with Panobinostat (Pano) for 1 and 6 h. mRNA expression was analyzed in triplicate in two biological replicates. ( N ) Western blot analysis of c-Myc (MYC) and ITGA6 protein expression in TOV-112D and OVSAHO PT-sen and PT-res clones treated or not with Panobinostat for 24 h. ( O ) Co-immunoprecipitation (Co-IP) analysis of c-Myc, HDAC1, and SP1 in TOV-112D PT-sen and PT-res cells treated or not with CDDP. Input shows the expression of the indicated proteins in the lysates used for IP experiments; IgG represents the control IP using an unrelated antibody. ( P ) Western blot analysis of ITGA6 expression in ITGA6 HIGH and ITGA6 LOW subpopulations treated with different HDACs inhibitors for 24 h (MS-275 preferentially inhibits HDAC1; Apicidin preferentially inhibits HDAC1 and HDAC3). In all western blots of the figure, GAPDH or Vinculin were used as loading control, as indicated. In ( C ), ( F ), and ( M ), mRNA expression was analyzed in triplicate and normalized to actin housekeeping gene and expressed in arbitrary units. In all the graphs of the figure, statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation.

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Luciferase, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Binding Assay, Generated, Clone Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Graph reporting the percentage of PT-sen and PT-res TOV-112D cells adhered on laminin 10 (LM10) coated plates. The percentage of ITGA6 positive cells in each PT-res clone is reported in the legend. ( B ) Graph reporting the percentage of PT-sen and PT-res TOV-112D cells adhered on LM10 in the presence of IgG (control) or of the specific anti-ITGA6 blocking antibody (GoH3). In ( A ) and ( B ), data represent the mean (± SD) of at least 3 replicates and BSA was used as negative control of cell adhesion. ( C ) Typical images of PT-sen and PT-res TOV-112D cells labeled with the green fluorescent marker DiO (green) and cultured on a monolayer of mesothelial cells for 24 h, in the presence or not of the anti-ITGA6 blocking antibody. Cells were then fixed and stained with Phalloidin (F-Actin, red) and TO-PRO3 (nuclei, blue). Scale bars, 50 μm. The right graph, reports the number (mean ± SD) of cancer cells/field of four independent evaluations. ( D ) PT-sen, PT-res ITGA6WT, and PT-res ITGA6KO TOV-112D cells adhered on LM10. Inset: western blot analysis reporting ITGA6 expression in the used cells. Data are the mean (± SD) of 2 independent experiments performed in triplicates. BSA was used as negative control for adhesion. ( E ) Tables reporting the IC50 and the confidence interval (CI) ( n = 5) of TOV-112D PT-sen, PT-res ITGA6WT and PT-res ITGA6KO cells plated on plastic or in 0.5% Matrigel and treated with increasing doses of CDDP for 72 h. Fisher’s exact test was used to calculate the global p value reported under the tables. ( F ) Graph (left) and representative phase-contrast images (right 20X objective) reporting the area of ovaryspheres formed by PT-sen, PT-res ITGA6WT and PT-res ITGA6KO cells treated or not with CDDP (5μM) for 24 h. ( G ) Graph (left) and representative phase-contrast images (right) reporting the area of ovaryspheres formed by PT-sen, PT-res ITGA6WT and PT-res ITGA6KO cells plated on a mesothelial cell monolayer. White dashed boxes in phase-contrast images highlight the areas magnified in the right panels. In ( F ) and ( G ), data represent the median ( ± SD) of two independent experiments performed in triplicate in which at least 5 randomly selected fields were analyzed. Scale bars, 50 μm. In ( A – D ) and ( F , G ). statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Error bars represent Standard Deviation. .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Control, Blocking Assay, Negative Control, Labeling, Marker, Cell Culture, Staining, Western Blot, Expressing, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Western blot analysis of the indicated proteins in whole lysates of TOV-112D PT-res cells plated on LM10 coated dishes in the presence of IgG (as control) or of GoH3 Ab. ( B ) Western blot analysis evaluating ITGA6 and Snail expression in whole lysates of TOV-112D PT-res ITGA6WT and ITGA6KO cells plated or not on LM10 coated dishes for 1 and 3 h. ( C ) Western blot analysis evaluating the expression of ITGA6 and Snail in TOV112D PT-res ITGA6WT and KO cells and ITGA6KO transfected with vectors expressing the two isoforms of ITGA6 (isoform A, A6A and isoform B, A6B). Whole lysates were analyzed at the indicated time points after LM10 adhesion. ( D ) Graph reporting the area of ovaryspheres formed by TOV-112D PT-res cells stably transduced with control shRNA (sh-ctrl) or Snail shRNAs and plated on a mesothelial cell monolayer. In the inset Western blot reports Snail expression in the used cells. Data represent the median (±SD) of three independent experiments performed in triplicate in which at least 150 randomly selected cells were analyzed. ( E ) Graph reporting the distance covered by the individual cells described in ( D ), in Matrigel evasion assay calculated starting from the edge of the drop (mean ± SD of three independent experiments in which at least 10 randomly selected fields were analyzed). In D and E, statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). ( F ) qRT-PCR evaluating the mRNA expression of Snail in TOV-112D PT-res cells plated on Poly-lysine (negative control) or on LM10 coated dishes. mRNA expression (mean ± SD) was analyzed in triplicate and normalized to actin housekeeping gene expression. ( G , H ) Western blot analysis evaluating the expression of ITGA6 and Snail in TOV112D PT-res WT and KO cells plated on LM10 for 1 and 3 h and treated or not with the proteasome inhibitor, MG132 alone ( G ) or with CHX ( H ). ( I ) Western blot analysis evaluating the expression of ITGA6 and Snail in TOV112D PT-res ITGA6WT cells plated on LM10 for 1 and 3 h and treated or not with MG132 or with the GSK3β inhibitor, LiCl. ( J ) Western blot analysis evaluating the expression of the indicated proteins in lysates of TOV-112D PT-res ITGA6WT and ITGA6KO cells plated or not on LM10 coated dishes for 1 and 3 h (NA = not adherent). ( K ) Western blot analysis evaluating the expression of the indicated proteins in lysates of TOV-112D PT-res ITGA6WT cells plated on LM10 coated dishes for 1 and 3 h in the presence or not of the pan-SRC inhibitor Saracatinib. ( L ) Schematic representation of obtained results. After adhesion to LM10, ITGA6 activates Lyn/Src pathway and regulates Snail protein stability to mediate invasion, adhesion, and stemness of tumor cells. In all western blots of the figure, GAPDH, Tubulin, or Vinculin were used as loading control, as indicated.

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Western Blot, Control, Expressing, Transfection, Stable Transfection, Transduction, shRNA, Two Tailed Test, Quantitative RT-PCR, Negative Control, Gene Expression

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Western blot analysis evaluating the expression of ITGA6 and CD63 (marker of extracellular vesicles) in conditioned medium (CM) and whole cell lysates of TOV-112D PT-res cells treated with CDDP for the indicated time points. ( B ) Western blot analysis to compare ITGA6 and CD63 expression in whole lysates (LYS), CM, exosomes (EXO) and exosomes-depleted CM (DEPR). ( C ) Western blot analysis of ITGA6 and CD63 in exosomes isolated from CM of the indicated cells treated with CDDP for 3, 6, and 9 h. ( D , E ) Graphs reporting the area of ovaryspheres formed by TOV-112D parental cells primed with exosomes ( D ) or CM ( E ) of TOV-112D PT-res ITGA6WT and KO cells and then plated on a mesothelial cells monolayer (NC = Not Conditioned). In ( E ), representative phase-contrast images were also reported under the graph. Scale bars, 50 μm. In ( D ) and ( E ), data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). ( F ) Western blot analysis evaluating the expression of the indicated proteins in whole cell lysates of TOV-112D PT-sen cells challenged for the indicated time points with the CM from TOV-112D PT-res ITGA6WT or ITGA6KO cells. NC = Not Conditioned. ( G ) Western blot analysis evaluating the expression of the indicated proteins in TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM from TOV-112D PT-res WT in the presence or not of the anti-ITGA6 blocking antibody, GoH3. ( H ) Western blot analysis evaluating the expression of the indicated proteins in TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM from TOV-112D PT-res WT cells in the presence or not of the Src inhibitor, Saracatinib. ( I ) Co-immunoprecipitation (Co-IP) analysis of ITGA6 and IGF1Rβ receptor in TOV-112D PT-res ITGA6WT or ITGA6KO cells. ( J ) Co-IP analysis of ITGA6 and IGF1Rβ receptor in TOV-112D parental cells incubated or not (NC) with the CM from TOV-112D PT-res WT in the presence or not of the anti-ITGA6 blocking antibody, GoH3. In ( H ) and ( I ), input shows the expression of the indicated proteins in the lysates used for the IP experiments; IgG represents the control IP using an unrelated antibody. ( K ) Table reporting the expression of the 7 proteins related to IGF1R pathway present in the used cytokine array. The mean fold ( n = 3 replicates) reports the ratio between the expression of each cytokine in the CM of PT-res ITGA6KO compared to that in the CM of PT-res ITGA6WT cells. Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). ( L ) Western blot analysis evaluating the expression of IGFBP6 in CM and whole lysates of TOV-112D PT-res ITGA6WT or ITGA6KO cells. ( M ) Western blot analysis of the indicated proteins in cell lysates of TOV-112D PT-sen cells incubated or not (NC) for the indicated times with the CM of TOV-112D PT-res ITGA6 KO cells in presence or not of the specific anti-IGFBP6 blocking antibody. In the figure GAPDH was used as loading control. .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Western Blot, Expressing, Marker, Isolation, Two Tailed Test, Incubation, Blocking Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Schematic representation of the experimental procedures used. Mesothelial cells (LPL) cells were incubated or not with CM of TOV-112D PT-res ITGA6WT or ITGA6KO cells for 16 h and then used for adhesion assays with TOV-112D PT-sen cells or for protein and mRNA expression analysis by western blot and qRT-PCR. NC = Not Conditioned. ( B ) Graph (left) and representative phase-contrast images (right) reporting the area of ovaryspheres formed by TOV-112D PT-sen cells plated on mesothelial cells monolayer primed as described in ( A ). Data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. ( C ) Western blot analysis evaluating the expression of IGFBP6 and IGF2 in mesothelial whole cell lysates treated as described in ( A ). ( D ) Western blot analysis evaluating the expression of the indicated proteins in whole cell lysates of mesothelial cells conditioned or not (NC) for the indicated times with CM of TOV-112D PT-res ITGA6KO cells in presence or not of the specific anti-ITGA6 GoH3 Ab. In ( C ) and ( D ), Tubulin was used as loading control. ( E ) Graph reporting the mRNA expression of IGF2 and LAMA5 (LM) in mesothelial cells incubated for the indicated times as described in ( A ). NC = Not Conditioned. Data are the mean (± SD) of 3 replicates. ( F ) Clinical history of patient #1 reporting the timeline of chemotherapy treatments and ascites collections. The amount of CA125 (in blood samples) and ITGA6 (in ascites) were reported in red and blue, respectively. ( G ) Graph (top) and representative phase-contrast images (bottom) reporting the area of ovaryspheres formed by TOV-112D PT-sen cells plated on mesothelial cells monolayer conditioned or not (NC) for 16 h with ascites samples described in ( F ), in the presence of the specific anti-ITGA6 blocking antibody GoH3, as indicated. Data represent the median (± SD) of three independent experiments performed in triplicate in which at least 70 randomly selected cells were analyzed. ( H ) Schematic representation depicting the role of ITGA6 in inducing a PT-resistant phenotype (increased invasion, adhesion, and stemness) both at cell-autonomous and non-cell-autonomous levels, leading to the formation of the pre-metastatic niche. Overexpression of ITGA6 allows a better adhesion of PT-res cells to the mesothelium. ITGA6 engagement induces stabilization of Snail and inhibition of IGFBP6 expression that promote invasion and stem-like behavior. On the other side secreted ITGA6 acts on PT-sen and mesothelial cells to activate the IGF1R pathway and amplify the pro-metastatic signals. ITGA6 KO, or its inhibition with blocking antibodies, prevents the activation of pro-metastatic metastatic pathways both in PT-res cells and in recipient cells and impairs IGF1R signaling by releasing the inhibition on IGFBP6 transcription leading to its overproduction. In ( B ) and ( G ), data represent the mean (± SD) of two independent experiments performed in triplicate in which at least 10 randomly selected fields were analyzed. Scale bars, 50 μm. In the figure statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Incubation, Expressing, Western Blot, Quantitative RT-PCR, Control, Blocking Assay, Over Expression, Inhibition, Activation Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A ) Schematic representation of the in vivo experimental procedures. NSG mice injected intraperitoneally (IP) with TOV-112D PT-res ITGA6WT ( n = 11) or ITGA6KO ( n = 11) cells, were randomly divided into two group and treated ( n = 6) or not ( n = 5) with CBDCA as indicated. Mice were sacrificed 30 days after the injection. ( B ) Graph reporting total number of tumors in mice described in ( A ), determined by macroscopic and pathological analyses (total tumor burden). ( C , D ) Radar ( C ) and Dot ( D ) plots reporting the distribution of abdominal metastasis ( C ) and the total number of organs infiltrated by cancer cells ( D ) in mice described in ( A ). In ( C ), black (WT cells) and red (ITGA6KO cells) lines indicate the number (values 0 to 5 for untreated and 0 to 6 for CBDCA-treated mice) of mice affected for each district, as indicated. Typical images of hematoxylin and eosin (H&E) staining of the showing liver metastasis in mice injected with ITGA6WT PT-res cells and treated as described in ( A ). 5X (scale bar = 200 μm) and 10X (scale bar = 50 μm) images of the same field are shown. White dashed boxes represent the magnified areas. ( E , G ) Western blot analysis evaluating the expression of ITGA6 and Snail in tumor masses ( E ) and IGFBP6 in the tumor masses ( F ) and ascites ( G ) of mice described in ( A ). Tubulin and Ponceau were used as loading controls. Mice 3 and 4 injected with ITGA6KO cells were treated with CBDCA. ( H ) Schematic representation of the in vivo experimental procedures using EOC PDX model. NSG mice injected IP with PDX OV218.3 ( n = 20), were randomly divided into four groups and treated or not ( n = 5) with the specific anti-ITGA6 blocking antibody ( n = 5), P5G10 ( n = 5), with CBDCA ( n = 5) or with the combination of both ( n = 5), according to the scheme. ( I ) Radar plots reporting the distribution of abdominal metastasis in mice injected intraperitoneally and treated as in ( H ), as determined by macroscopic and pathological analyses. Colored bold lines in each plot indicate the number (values 0 to 5) of mice affected for each district. ( J ) Typical images of H&E analyses of the omentum and lungs of mice described in ( H ). For each condition, 20X (for omentum, scale bar = 50 μm) and 40X (for lungs, scale bar = 20 μm) images are shown. Yellow dashed lanes in lung images highlighted the tumor metastasis. ( K ) Graph reporting total number of metastasis/mouse treated as indicated and determined by pathological analyses ( L ) Western Blot analysis of γH2AX in tumor cells isolated from ascites of mice described in ( H ). ( M ) Typical images of Snail (green) expression on peritoneal metastasis collected from mice described in ( H ) and evaluated by IF analyses (nuclei are in blue). White dashed lines indicate the boundary between tumor masses and the peritoneal wall. Scale bars = 20 μm. In ( B ), ( D ), and ( K ) Statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation. .

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: In Vivo, Injection, Staining, Western Blot, Expressing, Blocking Assay, Isolation, Two Tailed Test, Standard Deviation

Journal: EMBO Molecular Medicine

Article Title: Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer

doi: 10.1038/s44321-024-00069-3

Figure Lengend Snippet: ( A , B ) Graph reporting the volume of ascitic fluids ( A ) and of explanted macroscopically identified tumors of NSG mice injected intraperitoneally with PT-res ITGA6 WT ( n = 11) and KO cells ( n = 11) and treated ( n = 6) or not ( n = 5) with CBDCA 30 mg/kg 3 times per week for 2 weeks. ( C ) Typical images of mice described in ( A ) and ( B ). Red arrows indicated the presence of macroscopically visible tumors. ( D ) Clinical history reporting the timeline of surgery, chemotherapy treatments (in green) and ascites collection of EOC patient who donate her ascites to establish PDX OV218.3 (see Methods section). ( E , F ) Graph reporting the volume of ascitic fluids ( E ) and number of tumor spheroids ( F ) in NSG mice ( n = 5/group of treatment) injected with PDX OV218.3 and treated or not with the specific anti-ITGA6 blocking antibody P5G10, with CBDCA or with the combination of both according to the scheme reported in Fig. . ( G ) IGFBP6 mRNA expression in tumor cells in ascites of mice described in ( E , F ). mRNA expression was analyzed in triplicate and normalized to actin housekeeping gene expression. The mean (±SD) expression for each mouse is reported in the graph. ( H ) IF analyses evaluating the expression of pIGF1Rβ (green) and ITGA6 (red) on peritoneal metastases collected from mice treated as indicated (nuclei are in blue). White dashed lines indicate the boundary between tumor masses and the peritoneal wall. Yellow dashed boxes represent the areas magnified in the zoomed images, on the right. Scale bars = 20 μm. In the figure, statistical significance was determined by a two-tailed, unpaired Student’s t-test (Exact p values were reported on graphs). Bars represent Standard Deviation.

Article Snippet: The relative luciferase activity was calculated as the ratio of firefly luciferase to Renilla luciferase. pRc-a6A and pRC-a6B plasmids encoding for ITGA6 isoforms were a kindly gift of A. Mercurio, RSV-SP1 vector was from Addgene (#12098).

Techniques: Injection, Blocking Assay, Expressing, Gene Expression, Two Tailed Test, Standard Deviation