Mice, diet, and cell lines
Wild type female BALB/c mice and NSG mice were purchased from Charles River Laboratory (Wilmington, MA, USA). All experimental protocols followed NIH Guidelines and were approved by the Institutional Animal Care and Use Committees of University of Illinois at Chicago. All mice were used for experiments at age 6 to 8 weeks. According to the power analysis, where expected biologically significant differences are greater than 25% with a standard deviation of 20% utilizing an alpha of 0.05 and power of 0.8; at least six animals were placed in each experimental group. Doxycycline diet (TD.08434) was purchased from Envigo (Madison, WI, USA). CT26 murine and human HT-29 colorectal cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in DMEM culture medium (Life technology, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen, Waltham, MA USA). EGFR5, mouse EGFR with site mutations enabling Cetuximab binding [53] was very kindly gifted by Dr. Yang-Xin Fu. Cell lines were recently authenticated prior to in vivo use and tested for mycoplasma contamination. cDNA was cloned into XhoI sites of the pLVX-IRES-puro parental plasmid (Fig. 1A). After the pLVX-EGFR5 vector was identified through DNA sequencing, wtCT26 cells were transfected with the vector via lipofectamine 2000 and selected with puromycin (Fig. 1B). HT29 human CRC with high intrinsic expression of EGFR (Fig. 1C) was transfected with an inducible LIGHT expressing vector. The LIGHT vector was constructed by inserting the human LIGHT cDNA into BamHI/EcoRI of pLVX-TRE3G plasmid (Fig. 1A). Next, HT-29 cells were transfected with pTet3G DNA and pLVX-TRE3G-hLIGHT sequentially and selected with G418 and puromycin, respectively, to establish a HT-29hLIGHTi cell clone with high expression levels of LIGHT (Fig. 1C).
Antibodies, enzymes, reagents, and kits
Purified anti-mouse CD3 (145-2C11) and anti-mouse CD28 (37.51) mAbs were purchased from BD PharMingen (San Diego, CA). Donkey anti-human IgG PE, antiCD45 FITC, anti-CD8 Pecy7, anti-CD3 APC, anti-CD4 PE, anti-CD25 Pacific blue, anti-CD69 Percp-cy5.5, anti-CD49b Pecy7, anti-CD11c pacific blue, anti-CD80 PE, anti-CD86 APC, and Annexin V Apoptosis Detection Kit were purchased from eBiosciences (San Diego, CA, USA). Pacific blue conjugated anti-mouse CD4 (GK1.5) was purchased from Biolegend (San Diego, CA, USA). Anti-mouse LIGHT was purchased from R&D system (Minneapolis, MN, USA). Anti-mouse CD3 was purchased from Abcam (Cambridge, MA, USA) and HRP-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CalPhos™ Mammalian Transfection Kit, Lenti-X Tet-One Inducible Expression Systems, G418, and doxycycline were purchased from Clontech (Mountain View, CA, USA). Plasmid DNA purification kit was purchased from QIAGEN (Valencia, CA) and BD Bioscience (San Jose, CA, USA.), CellTrace™ Far Red Cell Proliferation Kit, CellTrace™ CFSE Cell Proliferation Kit, Lipofectamine® 2000 Transfection Reagent, puromycin, and Trizol reagent were purchased from Invitrogen (Waltham, MA USA). Cell titer 96 aqueous non-radioactive cell proliferation assay kit and CytoTox G1780 non-radioactive cytotoxicity assay kit were purchased from Promega (Madison, WI, USA). LTβR-Ig was kindly gifted by Dr. Yang-Xin Fu. Collagenase IV and DNase I were purchased from Sigma (St. Louis, MO, USA). Restriction endonucleases enzymes including MluI, PstI, BamHI, EcoRI, T4 DNA ligase, and DH5 alpha competent E. coli cells were purchased from New England Biolabs (Ipswich, MA, USA). Phospho-EGFR reactive to EGFR/EGFR5 (Y1068) (D7A5) (Catalog number:3777 s), AKT, and ERK mAbs were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β actin was bought from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-EGFR (Cetuximab) was bought from Eli Lilly (New York, NY, USA).
Identification of CT26EGFR and HT29hLIGHTi cell lines
1 × 106 of CT26EGFR cells were harvested and suspended in 100 µl PBS containing 0.5% fetal bovine serum, incubated with anti-EGFR at 1 µg/ml at 4 °C for 60 min and then incubated with anti-rabbit IgG-488 at 1:500. Cells were analyzed with a cyan ADP analyzer (Beckman Coulter, Brea, CA, USA) after multiple washes. Events were collected and analyzed using Flow Jo software (Tree star Incorporated, Ashland, OR USA) (Fig. 1B). HT29hLIGHTi cells (1 × 106) were harvested 24 h after incubation with or without doxycycline at 1 µg/ml and suspended in 100 µl staining buffer. Cells were incubated with anti-EGFR or incubated with LTβR-Ig at 1 µg/ml at 4 °C for 60 min, followed by incubation with anti-rabbit-488 or anti-human IgG-PE at 4 °C for 30 min. The cells were analyzed as above (Fig. 1C, D).
Stimulation of CT26EGFR and HT-29hLIGHTi cells with EGF and Western blot
CT26EGFR and HT-29hLIGHTi (4 × 105 cells/well) were seeded into 6 well plates on day 0, and then cells were serum starved overnight in 1% FBS RPMI 1640 medium on day 2. On day 3, the cells were stimulated with human EGF at 100 ng/ml at 37 °C for 5 and 15 min in plain RPMI medium. Cells were lysed with RIPA buffer and protein concentrations in the cell lysates were determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Cell lysates of 25 µg protein were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Blots were blocked for 1 h at room temperature in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) and 0.1% Tween 20. Membranes were incubated 2 h with specific antibodies, washed three times in PBS containing 0.1% Tween 20, and detected using HRP-conjugated goat anti-rabbit IgG or rabbit anti-mouse IgG. After three washes in PBS containing 0.1% Tween 20, signals were revealed by an enhanced chemiluminescence detection system (Amersham) and visualized by autoradiography.
Cross linking of anti-EGFR x anti-CD3 for murine and human BsAb generation
Mouse anti-CD3 and anti-EGFR (cetuximab) were cross-linked with the protein-protein crosslinking kit (Thermo scientific, catalog number: 23456. Rockford, IL, USA). Briefly, for the anti-CD3 maleimide activation, 5 mg purified anti-mouse CD3 (145-2C11) (ebioscience, San Diego, CA, USA) was dialyzed against PBS to thoroughly remove Tris or glycine overnight. 82.5 µl of the Sulfo-SMCC solution was added to 5.0 ml anti-CD3 solution and incubated for 30 min at room temperature. Non-reacted Sulfo-SMCC from the maleimide-anti-CD3 reaction mixture was removed through the equilibrated desalting column. For cetuximab sulfhydryl modification, 4.5 mL of polyclonal cetuximab containing 5 mg Protein (Millipore, Temecula, CA, USA) and 500 μL of Conjugation Buffer (10X) was added to the activated Immobilized Reductant and mixed thoroughly. The mixture was incubated for 20 min at 65 °C. After centrifuge, the supernatant containing sulfhydryl- cetuximab was mixed with maleimide-anti-CD3 in approximately equal molar amounts and incubated for 60 min at room temperature for crosslinking. Crosslinking of anti-EGFR x human anti-CD3 for human BsAb generation was performed using the same steps.
ATC purification, activation, and expansion
Murine T cell activation and expansion were performed in 6 well plated coated with anti-CD3 at 2 µg/ml and soluble anti-CD28 (2 µg/ml) plus mIL-2 (100 U/ml). 5 ml of T-cells at 2 × 106 cell/ml was seeded into a single well. On day 2, cells were passaged to a 10 cm dish, at 1 × 106 cells/ml containing 100 U/ml murine IL-2. On day 4, 10 ml of complete T cell medium with 100 U/ml murine IL-2 was added to the dish. Cells were harvested on day 5.
Blood collection and use of human blood products for research were conducted under Institutional Review Board approved protocols at University of Virginia, and signed consents were obtained from normal and patient donors. ATC were generated in RPMI and cryopreserved. 25 ml of blood containing 1000 U/ml heparin was placed into Ficoll-Hypaque with an equal volume of 1X PBS. The interface with mononuclear cells was transferred to new 50 ml conical tube. After two PBS washes, cells were suspended in RPMI 1640. 1–1.5 × 106 PBMC/mL in culture were activated with 20 ng/ml of anti-human CD3 (OKT3) and 100IU rIL-2. Cells were expanded for up to 14 days ± 3 days.
Assessment of the dual binding activity of BsAb anti-CD3 x anti-EGFR
To identify anti-CD3 x anti-EGFR BsAb binding, different doses (25, 50, 100, 200, 500, 1000 ng) of BsAb were added to 1 × 106 CT26EGFR5 cells and mouse splenocytes, respectively, and incubated at 4 °C for 1.0 h. 0.25 µl (200x dilution) anti-Armenian Hamster IgG –FITC or 0.2 µl anti-rabbit IgG-FITC (250x dilution) were added to splenocytes or CT26EGFR5 cells after washing and incubated at 4 °C for 30 min in one pot. Cells were analyzed using a cyan ADP analyzer (Beckman Coulter, Brea, CA, USA) after two subsequent washes. Events were collected and analyzed using Flow Jo software (Tree star Incorporated, Ashland, OR, USA).
To identify the human BsAb (anti-CD3 x anti-EGFR) the method and procedures are similar to the murine BsAb identification except that human T cells and human colorectal cancer cells (HT29) were utilized, and the secondary antibodies were anti-mouse-IgG-PE and anti-human-IgG-PE.
To further demonstrate that the BsAb binds simultaneously specifically to both CD3 T-cells and colon cancer cells, murine or human CD3+T cells and murine CT26EGFR5 or human HT-29 cells were labeled with cell tracker violet and far red, respectively. The labeled T cells (5 × 105) and CT26EGFR5 or HT-29 cells were be mixed at 5:1 (T: CT26EGFR5 or T:HT-29) and incubated with BsAb at 1 µg/ml 1 h at 4 °C in the presence or absence of excess amounts of anti-CD3 (10 µg/ml), anti-EGFR (10 µg/ml), or both antibodies. The amount of Violet+Far red+ cell clusters were analyzed by flow cytometry.
Arming activated T cell with BsAb
Murine CD3+ T cells were suspended with 2% FBS, 1xPS/AmpB, HEPES RPMI1640 at 10 × 106/ml and 200 ng/ml murine BsAb, the mixture was incubated at 4 °C for 1 h and then washed twice with T-cell medium before use. Predetermined optimal arming dose of human BsAb is 50 ng/million T-cells/ml. ATC were re-suspended at 5–106 T cells/ml in RPMI Medium 1640 with 10% FCS/FBS, 1% L-Glutamine and 2% penicillin/ streptomycin in 15–50 ml conical tube. BsAb and ATC were incubated for 15 min at room temperature, then washed twice with complete RPMI prior to use.
Migration and binding of BATs to micro-island organoids
CT26EGFR cells were stained in PBS at 1 × 106 cells/ml containing 1 µl/ml of far red at 37 °C for 20 min and then incubated in 5 times original staining volumes of 2% FBS RPMI 1640 for another 5 min. Pellets were re-suspended in complete medium to 0.67 × 106 cells/ml. 500 µm micro-island cultures were lithographically patterned and spaced 1200 µm apart as described previously [54]. 23,000–25,000 tumor cells are organized in 90 islands within each well. On the second day, ATC, BsAb armed ATC or a mix of anti-CD3 (500 µg/1 × 106 cells/100 µl) and anti-EGFR (500 µg/1 × 106 cells/100 µl) armed ATC were labeled with CFSE prior to being co-cultured with CT26EGFR and HT-29hLIGHTi micro-island organoids. HT29hLIGHTi human colon cancer cells were cultured in the presence or absence of doxycycline at 1 µg/ml for 24 h, used as HT29LIGHT and HT29 cells, respectively, and then harvested and stained with far red after which they were seeded. The cells were fixed with 300 µl of 4% paraformaldehyde after 1.0 h incubation at 37 °C and rinsed twice with PBS. Pictures were taken under fluorescence microscope at 40x and 100x.
Non-radioactive cytotoxicity assay
Cytotoxicity mediated by ATC or BATs to CT26 or CT26EGFR and HT-29 or HT-29LIGHTi cells was performed with CytoTox G1780 Non-Radioactive Cytotoxicity assay from Promega (Madison, WI, USA). In brief, cancer cells were plated in 96 well plates, followed 4 h later by effector cells (murine or human ATC and armed ATC) at 2.5 × 104, 5 × 104 or 1 × 105 cells/well overnight in 37 °C CO2 coculture. After cell lysis, supernatants were reconstituted in substrate mix and cytotoxicity signal quantified at 490 nm per manufacturer instructions.
Proliferation assay of BsAb-armed ATC
CT26, CT26EGFR5, and HT29LIGHTi (3 × 105 cells/well) were seeded into 6 well plates and cultured overnight. HT29hLIGHTi human colon cancer cells were cultured in the presence or absence of doxycycline at 1 µg/ml for 24 h and used as HT29LIGHT and HT29 cells, respectively. Murine or human anti-CD3 (500 ng/ml), anti-EGFR (500 ng/ml), murine or human ATC (activated T cells, 3 × 106 cells/well), murine or human armed ATC (3 × 106 cell/well) with anti-CD3 plus anti-EGFR, and murine or human armed ATC with BsAb (BAT) (3 × 106 cells/well) were added into the wells in triplicate. Cells were harvested and counted with trypan blue exclusion 42 h after coculture. The fold change of CT26/CT26EGFR, or HT-29/HT-29LIGHT cell numbers were calculated by comparison with the original seeded cell number of CT26 or CT26EGFR and HT29 or HT29LIGHT.
Human ATC or BATs proliferation assay in vitro
2 × 105 cells ATC or BATs were co-cultured with 20,000 rad irradiated 2 × 104 HT-29 or HT-29LIGHT cells in U-bottom 96 well plates for 96 h. 20 µl of MTS solution was added to each well containing 100ul of medium and absorbance was measured at 490 nm after 1–4 h incubation.
Mouse models of CT26EGFR colorectal cancer
5 × 106 CT26EGFR cells in 100 µL PBS were inoculated subcutaneously in the right flank of Balb/C mice. When the tumor size reached ~150 mm3, animals were randomized into for equal numbers of animals in each group, and each animal was adoptively transferred in a non-blinded fashion with one of four treatments: 100 µl of RPMI, ATC or BATs with 3000U murine IL2, via retro orbital vein injection or anti-EGFR via intraperitoneal injection for four dosages with 7 day intervals. Tumor volumes were calculated using the equation ½ (long diameter × short diameter [2]). One part of the tumor was fixed in 10% formalin for immunohistochemical staining and another part of the tumor was dissociated with collagenase IV. The acquired single cell suspension (1 × 106/sample) was stained with conjugated antibodies and analyzed with FACS. The tumor infiltrating lymphocytes were analyzed after tumor tissues were stained with murine anti-CD3 followed by conjugated goat ant-rabbit IgG immunofluorescence staining using Image J.
For the CT26EGFR rechallenge experiment, 14 days after last treatment, 5 × 106 CT26EGFR cells in 100 µL PBS were inoculated subcutaneously in the left flank of the mouse. The mice were sacrificed on day 13 after rechallenge.
Mouse models of HT-29hLIGHTi colorectal cancer
6-week-old NSG mice were randomly divided into 8 groups of at least 6 mice per group. 5 × 106 HT-29hLIGHTi cells in 100 µL PBS were inoculated subcutaneously in the right flank of NSG mice. When the tumor size reached to ~150 mm3, half of the animals were randomized to have LIGHT expression induced, as previously [18]. Treatments were now not blinded. Groups 1 and 5 were injected with 100 µl of RPMI containing 3000U hIL2 per mouse via retro orbital vein. Groups 2 and 6 were injected with 200 µg anti-EGFR intraperitoneally. Groups 3 and 7 received 20 × 106 human activated T cells in 100 µl RPMI containing 3000U hIL-2 via retro orbital vein. Groups 4 and 8 received 20 × 106 of BATs in 100 ul RPMI containing 3000 U hIL-2 via retro orbital vein. Each mouse was given 1 cycle of treatment every 7 days for 6 cycles. Mice were sacrificed 48 h after last treatment, and analysis was as in murine tumors.
Immunohistochemistry
Tumor tissues were collected at the indicated times, fixed in 10% neutral buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E) and murine anti-CD3 antibody (1:750) (Ab11169, Abcam, and Cambridge, MA, USA) per protocol. Photographs were obtained with an Olympus BX51 fluorescence microscope and images were acquired using an Olympus DP71 CCD camera and DP Controller Software (Olympus, Center Valley, PA, USA). Whole tumor quantification was performed with inForm® software and confirmed with manual blinded quantification according to our standard clinical pathology protocol for primary colon cancers. In brief, the tumor was reviewed at low power and an area with the most TIL or lymphocytes was identified. In this location, 10 consecutive 400X images were evaluated and only cells infiltrating between tumor cells were counted. They were counted in a blind fashion with the tumor margin considered a maximum width of 360 µm from the edge of the tumor [55].
Xenografts underwent antigen retrieval and then slides were subjected to sequential staining with CD8 antibody (RTU, clone C8/144B, Biocare Medical, Pacheco, CA), CD3 (1:100, clone SP7, Abcam, Cambridge, UK), and CD4 (1:100, clone EP204, Sigma Aldrich, St. Louis, MO). Blocking, detection, and DAPI staining were performed with reagents from OpalTM 4-color Automation IHC Kit (Akoya Biosciences, #NEL820001KT, Menlo Park, CA). A spectral library was prepared using human tonsil control slides and pan-CK antibody (Clone AE1/AE3, #M3515, Agilent, Santa Clara, CA). Spectra of all library components were obtained using Vectra 3.0 Automated Quantitative Pathology System (Akoya Biosciences). Stained samples were scanned at 20X on Vectra 3.0 instrument and the spectral library was used to spectrally unmix images in inForm® software (Akoya Biosciences) for visualization of each color and to assess quality of each image. Unmixed images were used to segment cells and train inForm® software to phenotype and count CD3+/CD8+ and CD3+/CD4+ T-cell populations. The data were exported and analyzed in R where T-cell population densities (number of positive cell/mm2) were calculated.
3-dimensional multiplex confocal microscopy
Conjugation of fluorescent dyes to primary antibodies and tumor tissue preparation for imaging was as previously described [56]. Briefly, frozen tumor tissues in optical cutting temperature (OCT) compound were thawed at room temperature. Samples were embedded in 2% agarose and sectioned with a vibrating microtome (600 µm thickness). The following primary antibodies were conjugated to specific DyLight (DyL) dyes (Thermo Fisher): anti-human EGFR (528 clone, BioXCell), anti-human CD8 (HIT8a clone, Thermo Fisher), anti-human CD4 (RPA-T4 clone, Biolegend), anti-human CD3 (HIT3a clone, Biolegend), and anti-human CD31 (WM59 clone, Biolegend). Individual tissue sections were stained overnight with one of two panels: Panel 1) DAPI, DyL488-anti-hEGFR, DyL550-anti-hCD8, and DyL680-anti-hCD3; Panel 2) DAPI, DyL488-anti-hEGFR, DyL550-anti-hCD4, and DyL680-anti-hCD3. Sections were made optically transparent by room temperature incubation in 20% and 80% fructose solutions. All samples were imaged with a Caliber ID RS-G4 upright confocal microscope under the same laser settings: 405 nm excitation and 450/70 nm emission filter for DAPI; 488 nm excitation and 520/44 nm emission filter for DyL488-hEGFR; 561 nm excitation and 600/52 nm emission filter for DyL550-hCD8 and DyL550-hCD4; 640 nm excitation and 700/75 m nm emission filter for DyL680-hCD3. Multiple high-resolution fields of view were captured throughout each section using a Zeiss 40X water immersion objective (NA1.1; WD510 µm). We implemented FIJI ImageJ (imagej.net/Fiji) functions to threshold fluorescence intensity, pinpoint double positive CD3+ CD8+ or CD3+ CD4+ human T cells and calculate cell counts. Processed images were manually checked for accurate counting. In order to obtain representative 3D tumor images, we stained tumor microsections with DAPI, DyL488-anti-hEGFR, DyL550-anti-hCD8, DyL594-anti-hCD3, and DyL633-anti-hCD31, and imaged using a Leica TCS SP8 confocal microscope with the following laser settings: 405 nm excitation and 420–455 nm emission filter for DAPI; 488 nm excitation and 495–530 nm emission filter for DyL488-hEGFR, 550 nm and 563–579 nm filter for DyL550-hCD8, 594 nm and 603–624 nm filter for DyL594-hCD3, 633 nm and 648–662 nm filter for DyL633-hCD31. We constructed 3D visualizations of the tumor microsection images using Imaris software (Bitplane).
Statistical analysis
All microscopic images and manual counts were performed in a blind fashion. Statistical analysis was performed on data that met the assumption of the test and with similar variance between groups that are being compared, with two tailed Student’s t-test. Error bars represent SEM. *P < 0.05, **p < 0.01, and ***p < 0.001. Statistical significance was considered for p-value < 0.05. Criteria to include samples with biologic variance within two standard deviation of the mean were pre-established. All statistical analysis was performed with Microsoft Excel®.
Kelly Ng of Sunset District legacy business (left) Wah Mei School speaks about the school’s programs at Sunset Mercantile’s Lunar New Year Market on February 6, 2022. Heritage photo.
Sunset Chinese Cultural District working group members receive Certificates of Honor from Supervisor Mar for their efforts promoting the Sunset Chinese Cultural District.
A section of the Sunset Chinese Cultural District booth featured a children’s fan-making activity from Wah Mei School. Heritage photo.
The Sunset Chinese Cultural District booth was adjacent to the Chinese Historical Society booth, which featured panels from their Chinese in the Sunset exhibition. Heritage photo.
Our table featured a one-page information sheet on the Sunset Chinese Cultural District, in both English and Chinese. Supervisor Mar’s wife, Cecilia Wong, provided the Chinese translation. Heritage photo.
