Journal: Annals of Translational Medicine

Article Title: Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression

doi: 10.21037/atm-22-2663

Figure Lengend Snippet: EYA3 was elevated in the CRC cancerous biopsies and CRC cells. (A) EYA3 mRNA levels in cancerous CRC biopsies and their adjacent noncancerous tissues (Control) (n=20). **, P<0.01. (B) IHC staining results of EYA3 in one representative group of cancerous CRC biopsy and its adjacent noncancerous tissue. Bars =100 μm. (C,D) EYA3 protein levels in three representative groups of cancerous CRC biopsies and their adjacent noncancerous tissues. GAPDH was used as the loading control. (C) Immunoblot result. (D) Quantified protein levels. **, P<0.01. (E) EYA3 mRNA levels in HCEC-1CT cells and seven CRC cell lines (HT55, HCA-24, HCT-15, HCT-116, HT-29, SW620, and T84). **, P<0.01 and ***, P<0.001. (F,G) EYA3 protein levels in CRC cells. GAPDH was used as the loading control. (F) Immunoblot result. (G) Quantified protein levels (comparing with HCEC-1CT). **, P<0.01 and ***, P<0.001. mRNA, messenger RNA; CRC, colorectal cancer; IHC, immunohistochemistry.

Article Snippet: Seven CRC cell lines (HT-29, HT55, HCT-15, HCT-116, HCA-24, SW620, and T84) and one noncancerous human colon epithelial cell line (HCEC-1CT) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Control, Immunohistochemistry, Western Blot

Journal: Annals of Translational Medicine

Article Title: Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression

doi: 10.21037/atm-22-2663

Figure Lengend Snippet: Hypoxia induced the expression of EYA3. (A,B) Protein levels of HIF-1α and HIF-2α in three representative groups of cancerous CRC biopsies and their adjacent noncancerous tissues. GAPDH was used as the loading control. (A) Immunoblot result. (B) Quantified protein levels. **, P<0.01 and ***, P<0.001. (C,D) Protein levels of HIF-1α, HIF-2α, and EYA3 in hypoxia-treated cells (treatment with hypoxia for 0, 3, 6, and 9 h). GAPDH was used as the loading control. (C) Immunoblot result. (D) Quantified protein levels. *, P<0.05 and **, P<0.01. (E) The mRNA levels of HIF-1α , HIF-2α , and EYA3 in the Control-KD, HIF-1α-KD, and HIF-2α-KD cells treated with or without hypoxia for 9 h. **, P<0.01 and ***, P<0.001. mRNA, messenger RNA; HIF-1α, hypoxia-inducible factor 1α; CRC, colorectal cancer; HIF-1α, hypoxia-inducible factor 1α; KD, knockdown.

Article Snippet: Seven CRC cell lines (HT-29, HT55, HCT-15, HCT-116, HCA-24, SW620, and T84) and one noncancerous human colon epithelial cell line (HCEC-1CT) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Control, Western Blot, Knockdown

Journal: Annals of Translational Medicine

Article Title: Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression

doi: 10.21037/atm-22-2663

Figure Lengend Snippet: Knockdown of SIX5 and p300 inhibited cell proliferation and invasion. (A,B) p300 and SIX5 mRNA levels in HCEC-1CT cells and seven CRC cell lines (HT55, HCA-24, HCT-15, HCT-116, HT-29, SW620, and T84). *, P<0.05 and **, P<0.01. (A) p300; (B) SIX5. (C) p300 mRNA levels in control-KD and p300-KD cells under HT-29 background. **, P<0.01. (D) SIX5 mRNA levels in control-KD and SIX5-KD cells under HT-29 background. **, P<0.01. (E) Cell viability of Control-KD, p300-KD, and SIX5-KD cells (HT-29 background) at different time points (days 0, 1, 2, 3, 4, and 5). *, P<0.05; **, P<0.01; and ***, P<0.001. (F) Invaded cell numbers of control-KD, p300-KD, and SIX5-KD cells (HT-29 background). ***, P<0.001. CRC, colorectal cancer; KD, knockdown.

Article Snippet: Seven CRC cell lines (HT-29, HT55, HCT-15, HCT-116, HCA-24, SW620, and T84) and one noncancerous human colon epithelial cell line (HCEC-1CT) were acquired from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Control

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Comparable HCT15 and COLO205 cell binding by 88hIgG1, 88mIgG3 and FG88.2 (hybridoma mAb)(A). Significantly reduced direct cytotoxicity (PI uptake) on HCT15 by 88hIgG1 compared to 88mIgG3 and FG88.2 (B). Significantly reduced proliferation inhibition by 88hIgG1 compared to 88mIgG3 and FG88.2 on COLO205 (C) and HCT15 (D). Significance (88hIgG1 compared to 88mIgG3) deduced from two-way ANOVA.

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Binding Assay, Inhibition

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Constant domain shuffling suggests no significant contribution by CH1 to direct cytotoxicity (PI uptake, HCT15, A; proliferation inhibition on COLO205, B). In contrast, CH3 (1m3 and 3h3) contributes significantly to direct cytotoxicity, with a minor contribution by mCH2 only evident in a loss-of-function approach (3h2) (PI uptake, HCT15, C; proliferation inhibition, COLO205, D). Significance versus the respective parental constructs was deduced from two-way ANOVA. Significantly increased avidity and decreased off-rate by 1m3; with significantly decreased avidity and increased off-rate by 3h2 and more pronounced by 3h3, confirming the major CH3 and minor CH2 contributions (E, F). Significance deduced using one-way ANOVA, with Dunnett’s corrections for multiple comparisons.

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Inhibition, Construct

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Subdomain divisions of CH2CH3 identified two regions that did not contribute: SD232-294 and SD390-447, as well as two regions that significantly contributed to direct cytotoxicity and avidity: SD286-345 (CH2) as well as SD339-397 (CH3). Significantly increased PI uptake by both constructs and the combination (SD286-397) compared to 88hIgG1 on COLO205 (A) and HCT15 (B). Significantly increased proliferation inhibition by both constructs and the combination compared to 88hIgG1 on COLO205 (C) and HCT15 (D). Significance (A – D) was deduced from two-way ANOVA. Significantly increased avidity (SPR), resulting mainly from reduced off-rates by SD286-345 and SD339-397, as well as the combination (SD286-397) (E and F, respectively). Significantly reduced CDC activity (HCT15) by SD339-397 compared to 88hIgG1 (G); maintenance of ADCC activity (COLO205) by the aforementioned constructs (H). Significance versus respective parental constructs (E-H) was deduced from one-way ANOVA, with Dunnett’s corrections for multiple comparisons.

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Construct, Inhibition, Activity Assay

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Significantly increased PI uptake (A) and proliferation inhibition (B) by SD339-378 compared to 88hIgG1 on HCT15. Significantly reduced proliferation inhibition by SD307-345 compared to SD286-345, suggesting a contribution by SD286-306 (C). Significantly increased proliferation inhibition by the combination of SD286-306+339-378 compared to 88hIgG1 on HCT15 (D) and COLO205 (E), as well as PI uptake on COLO205 (F). Significantly increased avidity (SPR) by SD339-378 as well as SD286-306+339-378 compared to 88hIgG1 (G). Maintenance of CDC activity on HCT15 (H) and ADCC on COLO205 (I) by SD339-378 as well as SD286-306+339-378 compared to 88hIgG1. Significance versus respective parental constructs was deduced from two-way ANOVA (direct cytotoxicity) or one-way ANOVA with Dunnett’s corrections for multiple comparisons(avidity, and effector functions).

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Inhibition, Activity Assay, Construct

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Reversion to human sequence of three residues in IEDB-predicted MHCII binding cluster 1 (Supplementary Fig. 2) created the lead candidate i88G1 (DI1). Significantly increased proliferation inhibition on HCT15 (A) and COLO205 (B) by i88G1 compared to 88hIgG1, now matching 88mIgG3 activity. Significance deduced from two-way ANOVA. DI2 displayed a consistent reduction in cytotoxicity compared to DI1 (A and B). Significantly increased avidity (SPR) by i88G1 compared to 88hIgG1, significantly decreased avidity by DI2 compared to 88mIgG3 (C), one-way ANOVA with Dunnett’s corrections for multiple comparisons. Significantly reduced, yet remaining subnanomolar, ADCC (COLO205, D) as well as, significantly improved CDC (HCT15, E) activity by i88G1 compared to 88hIgG1 (two-way ANOVA). Evidence of cellular detachment, aggregation and pore formation (white arrows point to irregular pores) by i88G1 on HCT15 (F).

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Sequencing, Binding Assay, Inhibition, Activity Assay

Journal: Cancer research

Article Title: Engineering the human Fc-region enables direct cell killing by cancer glycan-targeting antibodies without the need for immune effector cells or complement

doi: 10.1158/0008-5472.CAN-19-3599

Figure Lengend Snippet: Significantly increased proliferation inhibition (A) and PI uptake (B) on COLO205 by i129G1 compared to 129hIgG1. Direct cell killing of low to moderate binding cancer cell lines was negligible (Supplementary Fig. 4). Significantly increased functional affinity (SPR) by i129G1 compared to 129hIgG1 (C). i129G1 maintains nanomolar ADCC activity on COLO205, but with significantly reduced overall lysis compared to h129hIgG1 (D). Nanomolar CDC activity by i129G1 on COLO205 is significantly increased compared to 129hIgG1 (E). Evidence of cellular detachment, aggregation and pore forming ability by i129G1 on COLO205 using SEM, white arrows point to irregular pores (F). Significant in vivo tumor control by i129G1 compared to vehicle control and compared to 129hIgG1 in a COLO205 xenograft model (Balb/c nude mice) (G). Individual tumor growth curves are shown in Supplementary Fig. 5. No significant effect on mean body weight during the course of the mouse study (H). Dose-dependent binding of rhFcRn by i129G1 and 129hIgG1 at pH6.0 (I). Significantly increased binding by i129G1 compared to 129hIgG1 at the top two concentrations. Negligible binding at pH7.0 by both constructs (I). Similar SEC-MALS profiles for i129G1 compared to 129hIgG1(Ji). A small increase in higher MW species is evident in i129G1 compared to 129hIgG1 via AUC (Jii). No significant increase in C4d generation upon incubation with human serum by i129G1, compared to 129hIgG1 (Jiii). Significance versus respective parental constructs was deduced from two-way ANOVA (direct cytotoxicity, effector functions, rhFcRn binding, in vivo tumor control) or one-way ANOVA (functional affinity and C4d detection), with Dunnett’s corrections for multiple comparisons.

Article Snippet: Colorectal cancer cell lines (COLO205 and HCT15) as well as the murine myeloma NS0 cell line were purchased from ATCC (Virginia, USA).

Techniques: Inhibition, Binding Assay, Functional Assay, Activity Assay, Lysis, In Vivo, Construct, Incubation