Anti-HER2 induced myeloid cell alterations correspond with increasing vascular maturation in a murine model of HER2+ breast cancer

Virostko

et al. 2020

Preprint

Background: Trastuzumab, a clinical antibody targeted to the human epidermal growth factor receptor 2 (HER2), has been shown to sensitize cells to radiation in vitro. Current studies lack longitudinal evaluation of cellular response and in vivo data is limited. The purpose of this study is to quantify the effects of combination trastuzumab and radiation therapy in vitro and in vivo over time to determine if there is a synergistic interaction. Methods: EGFP expressing BT474, SKBR3 and MDA-MB-231 cell lines were treated with 0.1 ng/ml of trastuzumab, 5 or 10 Gy of radiation, or combination treatment, and imaged using fluorescence live cell microscopy for one week. The Bliss independence model was used to quantify the effects of combination treatment. HER2+ tumor bearing mice (female NU/J) (N=34) were treated with saline, 10 mg/kg of trastuzumab, 5 or 10 Gy of radiation, or combination treatment. Tumor size was measured three times per week for four weeks via caliper measurements. Additional mice (N=13) were treated with 10 mg/kg of trastuzumab, 5 Gy of radiation, or combination treatment. Tumors were harvested at one week and evaluated with immunohistochemistry for inflammation (CD45), vascularity (CD31 and α-SMA), and hypoxia (pimonidazole). Results: Altering the order of therapies did not significantly affect BT474 cell proliferation in vitro (P>0.05). The interaction index calculations revealed additive effects of trastuzumab and radiation treatment in all three cell lines in vitro. In vivo results revealed significant differences in tumor response between mice treated with 5 and 10 Gy single agent radiation (P < 0.001); however, no difference was seen in the combination groups when trastuzumab was added to the radiation regimen (P=0.56), indicating a lower dose of radiation could be used without decreasing therapeutic efficacy. Histology results revealed increases in inflammation (CD45+) in mice receiving trastuzumab (P<0.05). Conclusions: Longitudinal evaluation of cell proliferation in vitro showed additive effects of combination therapy. In vivo results show a potential to achieve the same efficacy of treatment with reduced radiation when also administering trastuzumab. Further evaluation of tumor microenvironmental alterations during treatment could identify mechanisms of increased therapeutic efficacy in this regimen.

Section: Discussionmentioning

confidence: 96%

Quantifying the effects of combination trastuzumab and radiation therapy in human epidermal growth factor receptor 2 positive breast cancer

Bloom

Virostko

et al. 2020

Preprint

“…Furthermore, multiple cytokine expression would be an interesting future direction to explore in an in vivo tumor model comparing CD4 T-cell. Biological validation of T-cell immune in ltration can be achieved through ow cytometry against peripheral blood [20,[47][48][49]. 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.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, FcgR-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].…”

Section: Introductionmentioning

confidence: 99%

CD4 T-cell immune stimulation of HER2+ breast cancer cells alters response to trastuzumab in vitro

Mansur

Dugger

et al. 2020

Preprint

Self Cite

Introduction: The HER2+ tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory 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 influence 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 or without trastuzumab (10, 25, 50 and 100 mg/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-a expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test. Results: The viability of HER2+ cancer cells significantly decreased when exposed to 25 mg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-a expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-a and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32).Conclusions: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-a receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2+ breast cancer, which has potential to reducing tumor burden.

“…Moreover, FcgR-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 associated with immune cell in ltration, there exists a lack of longitudinal studies that examine trastuzumab-induced CD4+ immune interaction with HER2+ breast cancer [20].…”

Section: Introductionmentioning

confidence: 99%

CD4 T-cell immune stimulation of HER2+ breast cancer cells alters response to trastuzumab in vitro

Mansur

Dugger

et al. 2020

Preprint

Self Cite

Introduction: The HER2+ tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory 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 to measure T-cell influence 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 or without trastuzumab (10, 25, 50 and 100 mg/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-a expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test.Results: The viability of HER2+ cancer cells significantly decreased when exposed to 25 mg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-a expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-a and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32).Conclusions: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-a receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2+ breast cancer, which has potential to reducing tumor burden.