CD4 T-Cell Immune Stimulation of HER2+ Breast Cancer Cells in Response to Trastuzumab In Vitro

Introduction: The HER2+ tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor inltrating lymphocytes, which produce pro-inammatory cytokines. Determining the effect of T-cells on HER2+ cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy imaging to measure T-cell inuence on trastuzumab in HER2+ breast cancer. Methods: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4+ T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with trastuzumab (10, 25, 50 and 100 (cid:0) g/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2+ breast cancer. HER2 and TNF- (cid:0) expression were evaluated with western blot and ELISA, respectively. Signicance was calculated using a two-tailed parametric t-test. Results: The viability of HER2+ cancer cells signicantly decreased when exposed to 25 (cid:0) g/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells signicantly increased TNF- (cid:0) expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF- (cid:0) and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.49). Conclusions: The presence of T-cells signicantly increases the ecacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. TNF- (cid:0) expression suggests cytokines may interact with trastuzumab-induced HER2 receptor blockade. Examining molecular mechanisms of breast cancer immune inltration has the potential to improve response to targeted therapies.


Introduction
Twenty-ve percent of newly diagnosed breast cancer cases will overexpress the human epidermal growth factor receptor 2 (HER2) gene [1]. Preclinical and clinical studies indicate that the immune microenvironment of HER2+ tumors is driven by tumor in ltrating lymphocytes and macrophages, which can produce pro-in ammatory cytokines [2][3][4]. Immune in ammatory cytokine expression has been demonstrated to in uence tumor progression and proliferation, highlighting the interplay between immune interaction, tumor phenotype and progression [2,3].
Trastuzumab is a clinically approved humanized monoclonal antibody that prevents the dimerization of the HER2 receptor [5,6]. Trastuzumab is a key component in the treatment of primary and metastatic HER2+ breast cancer, improving progression-free survival [7][8][9][10]. Trastuzumab has been observed to exhibit immunogenic qualities through increased granzyme release and natural killer cell activation [11][12][13][14][15][16][17]. Lee et al. used immunohistochemistry and found patients with increased tumor in ltrating leukocytes (TIL) responded more favorably to trastuzumab, suggesting that TILs may serve as a biomarker to identify which HER2+ breast cancer patients would most bene t from trastuzumab [16].
Moreover, Gagliato et al. found that patients with increased TIL were associated with decreased tumor recurrence [14]. Preclinically, trastuzumab has been observed to increase CD11c and F4/80 (markers of dendritic cell and macrophages, respectively) in in vivo models of HER2+ breast cancer, highlighting the immunogenic potential of anti-HER2 therapy [15]. Moreover, Fc R-mediated stimulation of CD4+ T-cells and activation of CD4+ T-cells with HER2-primed dendritic cell vaccines reduced tumor burden through tumor-speci c T-cell response [18,19]. Although clinical studies have shown successful trastuzumab therapy is dependent on immune cell in ltration, there exists a lack of longitudinal studies that examine trastuzumab-induced CD4+ immune interaction with HER2+ breast cancer [20].
Culturing of cancer cells with immune cells, or onco-immune co-culturing, has been used to study immune interactions between cancer cells and tumor associated macrophages (TAMs) [21][22][23]. Castellaro et al. co-cultured MCF-7 breast cancer cells with TAMs and found TAMs promoted cell proliferation and metastasis. Furthermore, Castellaro et al. found macrophages increased breast cancer's resistance to tamoxifen, highlighting the interaction between immune cell presence and response to therapy [21]. While onco-immune co-culturing has been studied with cancer cells and TAMs, to our knowledge, the impact of T-cells on HER2+ breast cancer and subsequent longitudinal response to anti-HER2 therapy has not been investigated.
The purpose of this study is to investigate T-cell in uence on HER2+ breast cancer in response to anti-HER2 trastuzumab therapy. This study used longitudinal live cell imaging to quantify the effect of immune cell presence on trastuzumab-treated HER2+ breast cancer through in vitro co-culturing of CD4+ T-cells and HER2+ cancer cell lines. This data has potential to serve as the foundation for future immune-

Fluorescence transfection of breast cancer cell lines
The SKBR3 cell line was transfected to express green uorescent protein (GFP). A GFP plasmid was cloned into a Sleeping Beauty compatible vector (Addgene plasmid #60525). The GFP plasmid was cotransfected with pCMV (CAT) T7-SB100 Sleeping Beauty transposase (Addgene plasmid #34879) with Lipofectamine LTX (Thermo Fisher Scienti c, 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 uorescence and cancer cell viability was determined by quantifying change in uorescence signal (see Supplemental Figure 1). In vitro experiments examining treatment response in HER2+ breast cancer cell lines, T-cell in uence 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 Scienti c. 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% con uence at the nal 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 uorescence images were collected every 3-6 hours using 10× magni cation (Excitation/emission: 440-480/504-544 nm for green channels and 565-605/625-705 nm for red channels). Phase con uence and uorescence 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 speci ed timepoints and percent change was determined by ((X 1 -X 0 )/X 0 ) 100, where X 0 and X 1 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 96well 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 ve additional days (see Fig.2). Percent change in cancer cell viability was normalized to initial con uence. Mean con uence was summarized by averaging con uence at speci ed timepoints and percent change was determined. Each treatment group has 4-8 replicates.
T-cell in uence 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 ve 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 Tcells through media change. On day 2, trastuzumab was removed through media renewal. Following trastuzumab treatment, cells were longitudinally observed for ve 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 uorescence images were collected every 6 hours using 20 magni cation (Excitation/emission: 470/525 nm for green channels). Images were analyzed with MATLAB image analysis code to quantify the number of uorescent objects per eld 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 ve additional days. Changes in the number of uorescent objects were used to determine changes in cancer cell viability. Each treatment group has 5-6 replicates.
HER2 and TNF-quanti cation BT474, SKBR3, MDA-MB-453, and MDA-MB-231 cancer cells were washed with cold PBS and lysed. Lysates were centrifuged and collected for quanti cation with a Nanodrop 2000c spectrophotometer (Thermo Fisher Scienti c, 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 con rm 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.
TNF-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 quanti ed with a Cytation5 microscope (BioTek Instruments, Winooski, VT). Samples were averaged and normalized to expression on day 0.

Statistical analysis
Cell viability in co-cultured groups was quanti ed through longitudinal quanti cation of cell uorescence (see Supplemental Figure 1). Groups were summarized by average con uence, average cell number ± standard error of mean (SEM). A Student's t-test was used to assess group differences. Correlation of con uence and uorescence was analyzed by computing the Pearson correlation coe cient. The Grubbs outlier test was used to eliminate any data points that were statistical outliers. A p-value < 0.05 was considered statistically signi cant. All data and gures were analyzed using GraphPad Prism 7 (La Jolla, CA, USA).

Results
Longitudinal in vitro imaging reveals trastuzumab effects are saturated at higher concentrations µg/mL of trastuzumab, a signi cant decrease in cell growth compared to control on day 7 was only observed in BT474 cancer cells (p < 0.01). In groups treated with 10 µg/mL of trastuzumab, BT474 cancer cells were observed to have a 74.0 ± 3.6% increase in cell growth on day 7 when normalized to baseline cell growth on day 0 (p < 0.01), SKBR3 cancer cells were observed to have a 452.5 ± 60.6% increase in cell growth on day 7 (p = 0.48), and MDA-MB-453 cancer cells were observed to have 313.4 ± 14% increase in cell growth on day 7 (p = 0.06). In groups treated with 25 µg/mL trastuzumab, BT474 cancer cells were observed to have 61.9 ± 4.8% increase in cell growth on day 7 (p < 0.01), SKBR3 cancer cells were observed to have 384.8 ± 29.9% increase in cell growth on day 7 (p = 0.03), and MDA-MB-453 cancer cells were observed to have 213.3 ± 16% increase in cell growth on day 7 (p < 0.01).
BT474 cells treated with 25 µg/mL trastuzumab were observed to have 61.9 ± 4.8% increase in cell growth on day 7, while those treated with 100 µg/mL trastuzumab were observed to have 58.3 ± 5.6% increase in cell growth (p = 0.38). SKBR3 cancer cells, cells treated with 25 µg/mL trastuzumab were observed to have 384.8 ± 29.9% cell growth on day 7, while those treated with 100 µg/mL trastuzumab were observed to have 380.5 ± 16% cell growth on day 7 (p = 0.72). MDA-MB-453 cancer cells treated with 25 µg/mL trastuzumab were observed to have 213.3 ± 16% cell growth on day 7, while those treated with 100 µg/mL trastuzumab were observed to have 211.2 ± 43% cell growth on day 7 (p = 0.89). Trastuzumab doses above 25 µg/mL were statistically similar (p > 0.05) showing no additional cytotoxic bene t, therefore 25 µg/mL was used for all future experiments.
T-cell co-culture increases trastuzumab e cacy in HER2+ breast cancer with respect to HER2 expression   5 displays BT474 cell viability in response to trastuzumab (25 µg/mL) with T-cells co-cultured on day 0 (BT474 co-culture) or on day 1 (BT474 delayed co-culture). When BT474 cells were treated with trastuzumab on day 1, BT474 cells demonstrated a 100.2% ± 3% increase in cell viability on day 7. When T-cells were introduced to BT474 simultaneously with trastuzumab treatment on day 1, BT474 cells demonstrated a 100.9% ± 6.8% increase in cell viability on day 7, which was statistically similar to BT474 cells treated with trastuzumab (p = 0.88). When BT474 cells were cultured with T-cells on day 0 and treated with trastuzumab on day 1, the cells demonstrated an 81.4 ± 7% increase in cell viability on day 7, a signi cant decrease in viability compared to BT474 cells treated with trastuzumab (p = 0.01). Trastuzumab treated SKBR3 cells co-cultured with T-cells exhibited a -9 ± 5.4% increase in cell viability and SKBR3 cells treated with trastuzumab and human recombinant TNF-exhibited a -11 ± 5.6% (p = 0.48).

Discussion
This study seeks to investigate immune stimulation of HER2+ breast cancer in response to anti-HER2 trastuzumab therapy and identify potential mechanisms of enhanced response. Through our experiments, it was observed that immune in uence on HER2+ breast cancer in response to anti-HER2 targeted therapy decreased cancer cell viability compared to cancer cells treated with trastuzumab without immune in uence. Our data shows immune response to trastuzumab treated HER2+ breast cancer is impacted by CD4+ T-cell stimulation. Delaying the timing of T-cell co-culture by introducing T-cells 24 hours after HER2+ breast cancer cells were seeded impacted the response of the cells to treatment. This signi cance was only observed in the cell lines highly overexpressing HER2, such as BT474 and SKBR3 (Fig.4). Slight differences in cancer cells treated with trastuzumab and co-cultured cells treated with trastuzumab were observed in HER2 moderately overexpressing cell line, MDA-MB-453; however, this difference was trending towards statistical signi cance, suggesting immune stimulation in trastuzumab treated HER2+ breast cancer cells is related to HER2 expression. HER2 overexpression has been noted to increase tumor resistance to hormone-based therapy and certain chemotherapies [26,27]; however, HER2 expression in relation to immune stimulation and trastuzumab response has not been characterized.
TNF-serves a dual role in tumor proliferation and apoptosis. TNF-has been shown to promote destruction of tumor vasculature and synergize with liposome-mediated chemotherapy [28][29][30]. Donato et al. used TNF-sensitive and TNF-resistant MCF-7 cell lines to study TNF-treatment on poly (ADP-ribose) polymerase (PARP) cleavage and cell death [30]. Increased PARP cleavage and cell death was observed in TNF-sensitive MCF-7 cells, compared to TNF-resistant MCF-7 cells, suggesting TNF treatment increased DNA damage and apoptosis [30]. Conversely, TNF-has also been observed to promote in ammation and tumor proliferation [31,32]. Egberts et al. used an invasion assay and found that pancreatic cancer cell lines treated with TNF-were observed to have increased invasive properties and invasion promoting proteins, such as interleukin -8 (IL-8) and matrix metallopeptidase-9 (MMP9) [31].
Similarly, increased expression of TNF-was proportional to tumor grade and increased expression of TNF-was observed in invasive breast ductal carcinomas. In our study, in vitro ELISA and longitudinal cell imaging suggest TNF-expression improves e cacy of anti-HER2 therapy. Treatment of cancer cells with TNF-and trastuzumab resulted in similar decreases in cell viability as trastuzumab-treated cancer cells co-cultured with T-cells in two HER2+ breast cancer cell lines.
TNF-expression has been studied in breast cancer response to therapy [33][34][35]. Lee et al. observed decreased cell viability in the MCF-7 cell line in response to TNF-therapy, hypothesizing that TNFdownregulates estrogen receptor expression [35]. While our study supports the role of TNF-in decreasing cell viability, two out of three HER2+ cell lines used in our study, SKBR3 and MDA-MB-453, do not overexpress estrogen receptors, suggesting cell viability was not exclusively the result of TNF-induced estrogen receptor downregulation. Mercogliano et al. used plasmid transfection to generate TNFoverexpressing HER2+ cell lines and found that TNF-overexpression correlated with trastuzumab resistance [34]. Differences in results from Mercogliano et al. and our study could be attributed to treatment incubation time and endpoint analysis. Mercogliano et al. measured changes in cell proliferation after 2 days post treatment, whereas our study measured treatment response up to ve days post treatment. Moreover, in our study, TNF-induced changes in cell proliferation occurred approximately 3.5 days after treatment.
While T-cell immune stimulation has been studied in preclinical models of cancer, these studies have focused on changes in CD4+ T-cell viability in the presence of cancer cells [36,37]. Zhu et al. used a MTT glucose metabolism assay to study CD4+ T-cell proliferation when co-cultured with SW480 colon cancer cells and observed that colorectal cancer cells increased apoptosis in lymphocytes [37]. Youssef et al. used Annexin-V staining to determine the impact of cancer cells and oxygenation on T-cell viability and found increased CD4+ T-cell apoptosis when co-cultured with cancer cells in hypoxic conditions, implying cancer cell signaling interaction with immune cells [36]. To our knowledge, no other study has characterized the effect of immune stimulation and anti-HER2 targeted therapies on breast cancer cell viability.
Limitations of this study include the lack of comparative data with CD8+ T-cells. CD8+ cytotoxic T-cells are observed to target cancerous tissue and patients with high CD8+ T-cell in ltration have a signi cant increase in survival [38]. TALL-104, a CD8+ T-cell cell line, can be used to study CD8+ T-cell in uence on trastuzumab treated HER2+ breast cancer [39,40]. An additional limitation is the lack of in vivo data detailing changes in CD4+ T-cell immune in ltration. Future in vivo studies may provide additional data and could potentially be simulated through engrafting HER2+ breast cancer in a humanized mouse.
Changes in immune in ltration can be determined through tracking of CD4+ T-cell localization with longitudinal noninvasive immuno-PET imaging [41,42]. Freise et al. used a [ 89 Zr] anti-CD4 cys-diabody to track CD4+ T-cell localization in vivo, with high uptake in the spleen, lymph nodes and thymus [41]. Biological validation of T-cell immune in ltration can be achieved through ow cytometry against peripheral blood [20,[43][44][45]. Future experiments could investigate the involvement of the Fc-region of CD4+ T-cells and whether co-precipitation experiments can identify interaction between CD4+ T-cells and HER2+ breast cancer cells treated with trastuzumab.

Conclusion
This study uses in vitro longitudinal live cell imaging and uorescent microscopy to track immune stimulation in trastuzumab treated HER2+ breast cancer. Our data produced preliminary evidence that TNF-receptor activation could interplay with HER2 receptor activity to affect overall cell viability. Importantly, information from this work can be used to provide insight into clinically relevant TIL in response to successful trastuzumab therapy, and can be used to identify mechanisms of enhanced response. Datasets generated in this study are available from the corresponding author upon reasonable request.
Code availability: Analysis code used in this study is available from the corresponding author upon request.  In vitro treatment response of HER2+ breast cancer to single agent trastuzumab. HER2+ breast cancer cells were treated with incremental doses of single agent trastuzumab therapy and longitudinal changes in cell con uence was observed over 7 days. In comparison to control groups, cancer cells treated with 25 µg/mL, 50 µg/mL and 100 µg/mL were statistically similar (p = 0.38, 0.72 and 0.89 in BT474, SKBR3 and MDA MB 453 cell lines, respectively) to one another and 25 µg/mL was used for subsequent experiments.