HER2+ breast cancer cell lines (BT474, SKBR3, MDA-MB-453), HER2- breast cancer cell line (MDA-MB-231), and CD4+ T-cell line (Jurkat) were obtained from ATCC (Manassas, VA, USA). BT474 cells were grown in improved minimal essential media (Invitrogen, Carlsbad, CA) with 10% FBS and 1% insulin. SKBR3 cells were grown in McCoy’s 5A media with 10% FBS and 2 mM L-glutamine. MDA-MB-453 cells were grown in Leibovitz’s L-15 media (Sigma, St. Louis, MO, USA) with 10% FBS. MDA-MB-231 cells were grown in Dulbecco’s minimal essential media (Gibco, Gaithersburg, MD, USA) with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate. Jurkat T-cells were grown in RPMI-1640 media (Gibco, Gaithersburg, MD, USA) with 10% FBS and 2 mM L-glutamine. Co-culturing of cancer cells and T-cells was conducted by suspending cancer cells and T-cells in cancer cell’s respective growth medium prior to plating.
Fluorescence transfection of breast cancer cell lines
The SKBR3 cell line was transfected to express green fluorescent protein (GFP). A GFP plasmid was cloned into a Sleeping Beauty compatible vector (Addgene plasmid #60525). The GFP plasmid was co-transfected with pCMV (CAT) T7-SB100 Sleeping Beauty transposase (Addgene plasmid #34879) with Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, USA). SKBR3 cells were selectively cultured with McCoy’s 5A media supplemented with 10% FBS, 1% L-glutamine and 200 µg/mL geneticin. Fluorescence activated cell sorting was used to separate cells and the highest 25% of GFP expressing cells were used in experiments. The pCMV (CAT)T7-SB100 plasmid and the pSBbie-Neo were gifts from Zsuzsanna Izsvak and Eric Kowarz, respectively [24, 25].
Live cell imaging and image analysis
Viable cells were engineered to express fluorescence and cancer cell viability was determined by quantifying change in fluorescence signal (see Supplemental Figure 1). In vitro experiments examining treatment response in HER2+ breast cancer cell lines, T-cell influence on HER2+ breast cancer response to trastuzumab, and evaluation of timing of T-cell introduction on trastuzumab treated HER2+ breast cancer were carried out for approximately 144 hours on 96-well glass bottom plates (Fisher Scientific. Catalog #165305) with an IncuCyte S3 imaging system (Essen Bioscience, Sartorius, Germany). Preliminary experiments were conducted to determine seeding density of cancer cells to facilitate longitudinal cell growth (BT474: 20,000 cells/well. SKBR3: 7,500 cells/well. MDA-MB-453: 25,000 cells/well. MDA-MB-231: 1,000 cells/well. Jurkat T-cells: 7,000 cells/well). Cell seeding densities that resulted in continuous exponential growth and ~80% confluence at the final imaging timepoint were used. To retain CD4+ T-cells in co-cultured wells during treatment, plates were centrifuged at 500 g for 5 minutes prior to treatment and drug removal. For imaging, phase contrast and fluorescence images were collected every 3-6 hours using 10× magnification (Excitation/emission: 440-480/504-544 nm for green channels and 565-605/625-705 nm for red channels). Phase confluence and fluorescence data was analyzed using the IncuCyte S3 Live-Cell Image Analysis System. Cells were counted by automated image analysis using background subtraction and brightness threshold (2 green calibrated units and 0.8 red calibrated units). Mean values were summarized by averaging replicates at specified timepoints and percent change was determined by ((X1-X0)/X0)･100, where X0 and X1 represent cell viability at baseline and cell viability at subsequent timepoints, respectively.
Evaluation of treatment response to HER2+ breast cancer cell lines in vitro
To quantify HER2+ breast cancer response to trastuzumab, cell lines were treated with trastuzumab and viability was assessed (Fig.1A). GFP BT474, GFP SKBR3, and FUCCI MDA-MB-453 cells were plated in 96-well plates. On day 1, cells were treated with trastuzumab (10, 25, 50 and 100 µg/mL). On day 2, trastuzumab was removed through media change and cells were longitudinally observed for five additional days (see Fig.2). Percent change in cancer cell viability was normalized to initial confluence. Mean confluence was summarized by averaging confluence at specified timepoints and percent change was determined. Each treatment group has 4-8 replicates.
T-cell influence on HER2+ breast cancer’s response to trastuzumab
To test whether T-cell co-culture affects cancer cell response to trastuzumab, HER2+ (BT474, SKBR3 and MDA-MB-453) and HER2- (MDA-MB-231) cells were co-cultured with T-cells on a 96 well plate. On day 1, cells were treated with 25 µg/mL trastuzumab. On day 2, trastuzumab was removed through media renewal and replaced with fresh media (without trastuuzmab). Following trastuzumab treatment, cells were longitudinally observed for five additional days. Changes in cancer cell viability of co-cultured cells was normalized to that of initial cell viability on day 0 and the fold change per replicate was correlated to HER2 western blot expression with a Pearson Correlation Test. Each treatment group has 4-8 replicates.
Evaluating timing of T-cell co-culture on HER2+ breast cancer’s response to trastuzumab
To determine whether timing of immune stimulation of HER2+ breast cancer cells in response to trastuzumab impacts longitudinal treatment response, the timing of when T-cells were introduced to BT474 cell culture was examined (Fig.1C). T-cells were co-cultured with BT474 cancer cells either during initial cell seeding on day 0 or day 1. On day 1, groups were treated with trastuzumab (25 µg/mL) and T-cells through media change. On day 2, trastuzumab was removed through media renewal. Following trastuzumab treatment, cells were longitudinally observed for five additional days. Changes in cell viability was normalized to initial cell viability. Each group has 3 replicates.
Evaluating TNF- effect on trastuzumab induced HER2 receptor blockade
Experiments evaluating tumor necrosis factor-alpha (TNF-) on trastuzumab induced HER2 receptor blockade were carried out for approximately 120 hours with an EVOS M7000 imaging system (ThermoFisher, Waltham, MA, USA). Phase contrast and fluorescence images were collected every 6 hours using 20 magnification (Excitation/emission: 470/525 nm for green channels). Images were analyzed with MATLAB image analysis code to quantify the number of fluorescent objects per field of view.
To determine whether TNF- cytokine expression impacts cancer cell viability during trastuzumab induced HER2 receptor blockade, cancer cells were treated with trastuzumab and TNF- and response was longitudinally monitored (Fig.1D). BT474 and SKBR3 cells were co-cultured with T-cells on a 96 well plate. On day 1, cells were treated with either 1) 25 µg/mL trastuzumab, 2) 100 ng/mL human recombinant TNF- (R&D Systems. Catalog #: 210-TA-005) or 3) 25 µg/mL trastuzumab + 100 ng/mL TNF-. On day 2, treatment was removed through media renewal. Following trastuzumab treatment, cells were longitudinally observed for changes in viability for five additional days. Changes in the number of fluorescent objects were used to determine changes in cancer cell viability. Each treatment group has 5-6 replicates.
HER2 and TNF- quantification
BT474, SKBR3, MDA-MB-453, and MDA-MB-231 cancer cells were washed with cold PBS and lysed. Lysates were centrifuged and collected for quantification with a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 20 µg of protein per cell line was run on a NuPAGE Bis-Tris gel and transferred to a PVDF membrane. The membrane was blocked, probed with HRP conjugated mouse anti-human β-actin overnight at 4° C and developed with Amersham ECL western blot detection system (GE healthcare, Buckinghamshire, UK). Membranes were developed and visualized with an SRX-101A Medical Film Processor (Konica Minolta Medical and Graphic, Inc., Shanghai, China). After β-actin was used as a control to confirm consistent protein levels, the membrane was stripped and probed with 1:1000 rabbit anti-human HER2/ErbB2 primary antibody (Cell Signaling Technology, Danvers, MA, USA. Catalog no. #2242) and 1:1000 rabbit anti-human TNF- primary antibody (Cell Signaling Technology, Danvers, MA, USA. Catalog no. #C25C1) overnight at room temperature. The membrane was washed, incubated with 1:2000 HRP conjugated goat anti-rabbit IgG secondary antibody (Cell Signaling Technology. Catalog no. #7074) for 1 hour at room temperature. The membrane was redeveloped and visualized for protein expression. Bands were analyzed with the Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA). Individual cell line HER2 and TNF- expression was normalized to β-actin expression.
BT474, T-cells, and a co-culture of BT474 and T-cells were longitudinally imaged (N = 3 wells per group) to correlate changes in cancer cell viability with TNF- expression. All experimental groups were either treated with 25 µg/mL trastuzumab or media (control) at t = 24 hours. Treatment was removed at t = 48 hrs. On days 0 and 7, supernatant was collected for ELISA analysis. A TNF- ELISA kit (LSBio. Catalog No. LS-F2557-1) was used to quantify TNF- expression. ELISA expression was quantified with a Cytation5 microscope (BioTek Instruments, Winooski, VT). Samples were averaged and normalized to expression on day 0.
Cell viability in co-cultured groups was quantified through longitudinal quantification of cell fluorescence (see Supplemental Figure 1). Groups were summarized by average confluence, average cell number ± standard error of mean (SEM). A Student’s t-test was used to assess group differences. Correlation of confluence and fluorescence was analyzed by computing the Pearson correlation coefficient. The Grubbs outlier test was used to eliminate any data points that were statistical outliers. A p-value < 0.05 was considered statistically significant. All data and figures were analyzed using GraphPad Prism 7 (La Jolla, CA, USA).