human colorectal cancer cell lines ht29  (ATCC)

Journal: bioRxiv

Article Title: Molidustat Targets a Synthetic Lethal Vulnerability in APC-Mutant Colorectal Cancer through GSTP1 and PHD2 Co-Inhibition

doi: 10.64898/2026.01.31.702998

Figure Lengend Snippet: A. Percentage of Cleaved Caspase 3/7 positive HT29 (top), and RKO (bottom) cells. Cells were treated with Molidustat for 48 hours at indicated concentrations, 10uM Staurosporine was used as a positive control (100% cell death). Mean + SEM is assessed by unpaired two tailed Student’s t-test, **p<0.01, (ns) non-significant. B. Representative images of Cleaved Caspase-3/7 signal in DMSO, Molidustat (90 μM), and Staurosporine treated cells. Scale bar: 300 μm. C. Representative Western Blot of PHD2 levels in HT29 cells. D. Percentage confluency of HT29 cells post-transfection with the indicated guide RNAs. E. Cleaved Caspase-3/7 signal in HT29 cells post-transfection with the indicated crRNAs. Mean + SEM is assessed by two-way ANOVA, *p<0.05. N = 3 biologically independent experiments.

Article Snippet: Human colorectal cancer cell lines HT29 and RKO were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, 16000044), 1% (v/v) penicillin-streptomycin (Gibco, 15140122), and 2 mM L-glutamine (Sigma-Aldrich, G7513).

Techniques: Positive Control, Two Tailed Test, Western Blot, Transfection

Journal: BMC Biology

Article Title: Increased prolactin levels in pregnancy affect colorectal cancer aggressiveness

doi: 10.1186/s12915-025-02500-8

Figure Lengend Snippet: Prolactin sensitizes HT29 cells to TRAIL-mediated apoptosis. A Representative flow cytometry plots of the HT29 cells treated with 0.1 to 1000 ng/mL of soluble TRAIL in static conditions, under FSS, after PRL pre-exposure, and in FSS combined with PRL pre-exposure. B Quantification of the cell viability after soluble TRAIL treatment (statistical analysis was performed comparing all groups to the static 0 ng/mL TRAIL dosage without PRL or FSS). C Quantification of the TRAIL sensitization by each of the experimental groups (statistical analysis was performed comparing all groups to the 0.1 ng/mL TRAIL group without PRL or FSS). D Representative flow cytometry plot and quantification of cell viability, apoptosis, and necrosis of the HT29 cells treated with the DA liposomes in whole blood under FSS conditions. E Representative flow cytometry plot and quantification of cell viability, apoptosis, and necrosis of the PRL pre-exposed HT29 cells treated with the DA liposomes in whole blood under FSS conditions. Statistical significance was evaluated by either an ordinary one-way ANOVA or a two-way ANOVA and is shown as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 ( n = 3 biological replicates)

Article Snippet: The human colon adenocarcinoma cell lines HT29 (HTB38, ATCC) and COLO320DM (CCL-220, ATCC) were used throughout the study.

Techniques: Flow Cytometry, Liposomes

Journal: Molecular Cancer Therapeutics

Article Title: The Recycling Collagen Receptor uPARAP Is a Unique Mediator of Stromal Drug Delivery to Carcinoma Cells

doi: 10.1158/1535-7163.MCT-25-0051

Figure Lengend Snippet: Subcutaneous xenograft models with human lung and colon carcinoma cells recapitulate the uPARAP expression patterns found in human tumors. A and B, Human EBC-1 lung carcinoma cells and HT29 colon carcinoma cells are negative for uPARAP expression in vitro . EBC-1 and HT29 cells, as well as human SaOS-2 osteosarcoma cells (positive control), were cultured in vitro . A, (Top) Analysis of uPARAP expression by Western blotting. The uPARAP band (apparent relative molecular mass ∼180,000) is absent in the two carcinoma cell lines but present in the SaOS-2 cells. (Bottom) A Coomassie-stained SDS-PAGE gel with the same loading shows a uniform content of total protein in the three samples. B, Flow cytometry analysis of the same cell types following incubation with fluorescently labeled anti-uPARAP antibody, 2h9-AF647, or isotype control antibody, IgG-AF647. A prominent uptake of 2h9-AF647 was seen in the SaOS-2 cells, whereas no signal was observed in HT29 or EBC-1 cells. C, EBC-1 and HT29 tumors have infiltrating CAFs with strong expression of uPARAP. EBC-1 cells or HT29 cells were injected subcutaneously into CB17 SCID mice as in Supplementary Fig. S5. Tumor specimens from the mice were excised at predefined endpoints (see “Materials and Methods”) and immunostained for uPARAP. Tumor cells are uPARAP-negative, whereas infiltrating fibroblasts are uPARAP-positive (arrows in right figures at high magnification). Scale bars, 500 µm (low mag); 50 µm (high mag). D and E, CAF-directed uptake of uPARAP-directed mAb after administration in vivo . Tumor-bearing mice were injected intravenously with AF647-conjugated anti-uPARAP mAb, followed by flow cytometric analysis after 24 hours. D, Analysis of tumor material from mice with the indicated subcutaneous tumors. Tumors were excised, disaggregated into single-cell suspensions, and analyzed by flow cytometry, using markers for human cells (HLA and hCD29) and for activated fibroblasts (FAP), in addition to recording the fluorescence of the AF647-conjugated anti-uPARAP. Tumor cells are uPARAP-negative, whereas a pronounced uptake of the anti-uPARAP mAb is observed on infiltrating CAFs. EBC-1, n = 6. HT29, n = 3. See Supplementary Fig. S9 for fluorescence minus one panels. E, Uptake of uPARAP-directed mAb in normal tissues. (Left) Various organs from the EBC-1 tumor–bearing mice shown in D were excised and analyzed as in D , along with the tumor material. For the normal tissues, gating was performed on the specific cell population with the highest uPARAP signal, identified using additional markers (see Supplementary Fig. S10). For each tissue, the mean fluorescence intensity (MFI) of the cell population with the most prominent anti-uPARAP mAb uptake was compared with that of the CAFs (tumor). (Right) Fluorescence signal (MFI of AF647 fluorophore) of anti-uPARAP antibody 2h9 (2h9-AF647) or isotype control antibody (IgG-AF647) in the liver, after i.v. injection of mice in the same manner. n = 3 for all tissues except for the liver with IgG-AF647, where n = 4.

Article Snippet: The following cell lines were purchased from the indicated cell banks or commercial suppliers with designations as specified: human osteosarcoma SaOS-2 cell line (RRID:CVCL_0548, European Collection of Animal Cell Cultures; ECACC 89050205), human squamous cell lung carcinoma EBC-1 cell line (RRID:CVCL_2891, JCRB Cell Bank; JCRB0820), human osteosarcoma 143B cell line (RRID:CVCL_2270, ATCC; CRL-8303), human colorectal adenocarcinoma HT29 cell line (RRID:CVCL_A8EZ, ATCC; HTB-38), and luciferase-transfected HT29 (Luc-HT29) cells (RRID:CVCL_5J00, Revvity; IVISbrite HT29 Red F-luc, BW124353 ). uPARAP-deficient 143B cells were generated using CRISPR/Cas9-mediated gene editing as described ( , ).

Techniques: Expressing, In Vitro, Positive Control, Cell Culture, Western Blot, Staining, SDS Page, Flow Cytometry, Incubation, Labeling, Control, Injection, In Vivo, Fluorescence

Journal: Molecular Cancer Therapeutics

Article Title: The Recycling Collagen Receptor uPARAP Is a Unique Mediator of Stromal Drug Delivery to Carcinoma Cells

doi: 10.1158/1535-7163.MCT-25-0051

Figure Lengend Snippet: Targeting of uPARAP-negative tumors with a uPARAP-directed ADC. A and B, EBC-1 and HT29 carcinoma cells are insensitive to a uPARAP-directed ADC in vitro but sensitive to the free cytotoxin. A, EBC-1 cells (left), HT29 cells (middle), or uPARAP-expressing SaOS-2 cells (positive control; right) were cultured in vitro in the presence of the indicated concentrations of uPARAP-targeted ADC (9b7-MMAE) or nontargeted control ADC (aTNP-MMAE). After 72 hours of culture, the surviving cell fraction was determined by MTS assay. B, EBC-1 or HT29 cells were cultured in the presence of the indicated concentrations of uncoupled MMAE, after which the surviving cell fraction was determined as in A . C–E, Treatment of EBC-1 and HT29 tumors with 9b7-MMAE in vivo . EBC-1 or HT29 cells were injected into CB17 SCID mice as in Supplementary Figs. S2 and S5. Tumors were allowed to grow until reaching a size of 80 to 150 mm 3 , after which treatment was initiated with tail i.v. injections of the indicated doses of uPARAP-targeted 9b7-MMAE, nontargeted control ADC (aTNP-MMAE), unconjugated mAb 9b7 ( C only), or vehicle alone. Treatment was performed on days 0, 4, 7, and 11 (dotted lines). Tumor growth and regression were followed by palpation. For weight curves, see Supplementary Fig. S2. (Left) Tumor volume average ± SD for each treatment arm, ending the curve when the first mouse reached the humane endpoint. (Middle) Tumor volume in individual mice. (Right) Kaplan–Meier curves showing mouse survival for each treatment arm. ns, nonsignificant; P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by log-rank test with post hoc Bonferroni correction. C, n = 10 (PBS) and n = 9 (9b7 unconjugated, aTNP-MMAE, 9b7-MMAE). D and E, n = 8.

Article Snippet: The following cell lines were purchased from the indicated cell banks or commercial suppliers with designations as specified: human osteosarcoma SaOS-2 cell line (RRID:CVCL_0548, European Collection of Animal Cell Cultures; ECACC 89050205), human squamous cell lung carcinoma EBC-1 cell line (RRID:CVCL_2891, JCRB Cell Bank; JCRB0820), human osteosarcoma 143B cell line (RRID:CVCL_2270, ATCC; CRL-8303), human colorectal adenocarcinoma HT29 cell line (RRID:CVCL_A8EZ, ATCC; HTB-38), and luciferase-transfected HT29 (Luc-HT29) cells (RRID:CVCL_5J00, Revvity; IVISbrite HT29 Red F-luc, BW124353 ). uPARAP-deficient 143B cells were generated using CRISPR/Cas9-mediated gene editing as described ( , ).

Techniques: In Vitro, Expressing, Positive Control, Cell Culture, Control, MTS Assay, In Vivo, Injection

Journal: Molecular Cancer Therapeutics

Article Title: The Recycling Collagen Receptor uPARAP Is a Unique Mediator of Stromal Drug Delivery to Carcinoma Cells

doi: 10.1158/1535-7163.MCT-25-0051

Figure Lengend Snippet: Treatment of Luc-HT29 tumors in mouse bones. A, Histologic appearance of invasive bone tumors resulting from the homing of Luc-HT29 cells from the circulation. Mice were subjected to intra-CA injection of 1 × 10 6 Luc-HT29 cells on day 0 (see “Materials and Methods”; Supplementary Fig. S7). Thirteen to sixteen days after tumor cell injection, mice were sacrificed, and bones were excised and prepared for histology. Parallel sections were stained with hematoxylin and eosin (H&E), with a species-specific antibody against human lamin-B1 (staining of human-derived cancer cells), or with a uPARAP-specific antibody. Scale bars, 500 µm; 250 µm in the “zoom in” panel to the right. uPARAP-positive host cells are abundant in the area surrounding the tumor (examples shown with arrowheads) and are closely aligned with the tumor border (“zoom in” panel). B and C, Bone tumor growth in treated mice. Six days after the injection of Luc-HT29 cells as in A , treatment of mice was started by tail i.v. injection, using doses of 6 mg/kg (unconjugated mAb 9b7 or ADCs). Mice received a total of three doses on days 6, 9, and 12 (dotted vertical lines in C ). Tumor growth was followed and quantified by IVIS-based monitoring. For weight curves, see Supplementary Fig. S2. B, Examples showing IVIS-based tumor imaging for mice treated with PBS and uPARAP-targeting ADC, respectively. C, (Left and middle) Bone tumor signal obtained from the IVIS imaging; note the logarithmic scale for the representation of the IVIS signal. The Y -axis lower limit (1 × 10 5 p/second) corresponds to the total flux recorded in a non-luciferin injected mouse (background). Mice were euthanized at total flux > 1 × 10 9 (single leg). (Left) Average signal in treatment groups ± SD, ending the curve when the first mouse reached the humane endpoint. (Middle) Signal in individual mice. (Right) Survival curves of the four treatment arms; ns, nonsignificant; P > 0.05; ****, P < 0.0001 by log-rank test with post hoc Bonferroni correction. For one mouse in the 9b7-MMAE–treated group, tumor regrowth status was uncertain as an initial apparent increase in signal (days 41–48) was followed by a more stable signal (days 52–66) and a dramatically decreased luminescence signal at day 68 (termination of study). n = 8 (PBS, 9b7 unconjugated) and n = 9 (9b7-MMAE, aTNP-MMAE). D, Seeding and growth of additional tumors. (Left) Example with IVIS image showing tumors in the thorax region. These tumors were observed in 7, 4, 7, and 6 mice, respectively, in the groups treated with PBS, uncoupled mAb 9b7, aTNP-MMAE, and 9b7-MMAE. (Middle and right) IVIS-based growth curves of these tumors in all treatment groups. Representation of data is as in C .

Article Snippet: The following cell lines were purchased from the indicated cell banks or commercial suppliers with designations as specified: human osteosarcoma SaOS-2 cell line (RRID:CVCL_0548, European Collection of Animal Cell Cultures; ECACC 89050205), human squamous cell lung carcinoma EBC-1 cell line (RRID:CVCL_2891, JCRB Cell Bank; JCRB0820), human osteosarcoma 143B cell line (RRID:CVCL_2270, ATCC; CRL-8303), human colorectal adenocarcinoma HT29 cell line (RRID:CVCL_A8EZ, ATCC; HTB-38), and luciferase-transfected HT29 (Luc-HT29) cells (RRID:CVCL_5J00, Revvity; IVISbrite HT29 Red F-luc, BW124353 ). uPARAP-deficient 143B cells were generated using CRISPR/Cas9-mediated gene editing as described ( , ).

Techniques: Injection, Staining, Derivative Assay, Imaging