Temporal Changes in Immune Responses within the Tumor Microenvironment in the 4T1.2-HER2 Mammary Tumor Model

Background: The murine 4T1.2 triple-negative breast cancer model is widely used, but is poorly immunogenic with no dened tumor-associated antigens. A modied 4T1.2 model has been developed that stably expresses a surrogate tumor antigen, human epidermal growth factor receptor-2 (HER2). The goal of the current study was to characterize host immune responses in the 4T1.2-HER2 tumor model, focusing on the tumor microenvironment (TME) during the early stage of tumor development. Methods: Female BALB/c mice were orthotopically inoculated with 4T1.2-HER2 tumor cells and sacriced at day (D) 6, 9, 12, 15 and 18 post tumor inoculation. The phenotype and function of tumor-inltrating immune cells were assessed. Results: 4T1.2 and 4T1.2-HER2 tumor cells had similar proliferation rates in vitro. In contrast to the rapid progression of the parental 4T1.2 model, the 4T1.2-HER2 model demonstrated initial tumor growth followed by spontaneous tumor regression by D18 post tumor inoculation, which was not observed in scid mice. Following tumor regression, mice demonstrated either a second phase of tumor outgrowth or complete tumor rejection. Within the TME, the percentage of T cells was reduced at D9 and increased during tumor regression through D18 (p<0.05), whereas the percentage of myeloid-derived suppressor cells (MDSCs) increased during the initial tumor growth and was reduced by D18 (p<0.01). There was a stepwise increase in the percentage of IFNg + , IL-2 + and perforin + T cells and NK cells peaking at D12-15. Furthermore, tumor regression occurred concurrently with HER2-specic IFNg production from tumor-inltrating immune cells at D12 and D15 (p<0.05). During the second phase of 4T1.2-HER2 tumor growth, tumor volume was negatively correlated with immune inltration (r=0.662, p=0.052). Conclusions: These results suggest that the integration of a surrogate tumor antigen, human HER2,


Background
Breast cancer is the most commonly diagnosed cancer and one of the leading causes of death among women worldwide (1). The role of immune system in cancer control is well established and is achieved by a wide variety of anti-tumor mediators. CD8 + cytotoxic T cells (CTLs) and natural killer (NK) cells are primarily responsible for the killing of transformed cells, whereas CD4 + helper T cells orchestrate diverse immune responses by producing cytokines such as IFNγ (2,3). As tumor progression occurs, however, immunosuppressive cells from both the myeloid [myeloid-derived suppressor cells (MDSCs)] and lymphoid [regulatory T cells (Tregs)] lineages are also induced and recruited to the tumor microenvironment (TME) to facilitate immune escape (2)(3)(4)(5). The TME can be broadly categorized into "hot" or "cold" based on immune in ltration, which is closely related to prognosis and therapeutic e cacy (6)(7)(8). Breast cancer is traditionally viewed as non-immunogenic with relatively low mutation rates and tumor-in ltrating lymphocyte (TIL) levels. However, among the heterogeneous breast cancer subtypes, human epidermal growth factor receptor-2 (HER2)-positive and triple-negative breast cancer (TNBC) have higher levels of mutational burden and TIL in ltration compared to hormone receptor (HR)-positive breast cancer (9,10). Moreover, higher TIL levels are associated with reduced risk of tumor recurrence and mortality in HER2 + breast cancer and TNBC, and are predictive of response to neoadjuvant chemotherapy in all subtypes of breast cancer (11)(12)(13)(14).
In recent years, signi cant clinical advances have been made in cancer immunotherapies. T cells have been the major focus of therapeutic efforts due to their capacity to speci cally recognize tumor antigens derived from all cellular compartments, followed by direct killing of tumor cells and/or integration of various effector mechanisms (15). In particular, antibody blockade of immune checkpoints has demonstrated the most success in multiple cancer types including breast cancer (9,16,17). However, patient response rates to immune checkpoint blockade in most cancer types are limited to 10-25%, and a substantial percentage of patients eventually demonstrate disease progression after initial response to therapy for reasons that are not clearly understood (15,(18)(19)(20). Therefore, additional studies in preclinical cancer models that closely mimic human diseases are needed to better understand the mechanisms underlying cancer resistance to immunotherapies and to develop novel therapeutic strategies to improve clinical outcomes.
The 4T1.2 murine breast cancer model is a highly aggressive model of advanced TNBC. When the 4T1.2 tumor is inoculated orthotopically into the mammary fat pad, it spontaneously metastasizes to the lung and bone, two common sites of metastasis in breast cancer patients (21)(22)(23). Moreover, the use of this syngeneic model in immunocompetent hosts enables the study of the TME under the in uence of an intact immune system. Despite its unique strength in mimicking human metastatic breast cancer, the 4T1.2 model is poorly immunogenic with no de ned tumor-associated antigens (TAAs). This severely limits its utility in studying the effects and mechanisms of immunotherapeutic interventions on modulating antigen-speci c immune responses.
Moreover, s.c. 4T1.2-HER2 tumor growth demonstrates a spontaneous regression period which is dependent on CD8 + T cells (26). These data suggest that the addition of human HER2 protein to the 4T1.2 tumor may enhance its immunogenicity and thus enable the study of antigen-speci c immune responses in this clinically relevant breast cancer model. However, previous data on host immune responses in the 4T1.2-HER2 model in immunocompetent mice are limited, and are based on s.c. 4T1.2-HER2 tumor model which may not represent the mammary tissue environment in human breast cancer (26). Therefore, the goal of the current study was to characterize host immune responses in the orthotopic (intramammary inoculation) 4T1. In vitro cell proliferation assay  Table S1) or the corresponding isotype control antibodies for 30 min at 4°C. Following antibody incubation, cells were washed twice and xed with BD Cyto x™ xation buffer (BD Biosciences). All ow cytometric analyses were performed on a BD LSR-Fortessa (BD Biosciences) ow cytometer. Data analyses were preformed using FlowJo V10 (Tree Star, Ashland, OR).

Animal model
Female BALB/c and BALB/c scid mice were purchased from Jackson Laboratory (Bar Harbor, MA). In one experiment, BALB/c mice were orthotopically inoculated with 5x10 4 4T1.2 cells or 2x10 6 4T1.2-HER2 cells (n = 4-5/group) into the fourth mammary fat pad to compare tumor growth patterns in the 4T1.2 vs.
4T1.2-HER2 model. Additionally, BALB/c scid mice (n = 5) were orthotopically inoculated with 5x10 5 4T1.2 HER2 cells to evaluate the role of the adaptive immune system in regulating 4T1.2-HER2 tumor growth. In a second experiment, BALB/c mice were orthotopically inoculated with 2x10 6 4T1.2-HER2 cells and sacri ced at day 6, 9, 12, 15 and 18 post tumor inoculation (n = 8/time point) to evaluate immune outcomes during the early stage of 4T1.2-HER2 tumor development. In a third experiment, BALB/c mice were orthotopically inoculated with 5x10 5 , 1x10 6 or 2x10 6 4T1.2 HER2 cells (n = 4-5/group) and sacri ced between day 44-64 post tumor inoculation to evaluate tumor recurrence and immune outcomes during the second phase of tumor growth or tumor rejection. Primary tumor growth was measured three times/week using a digital caliper, and tumor volume was calculated following the equation V=(short 2 ×long)/2. All mice were housed at the Pennsylvania State University and maintained on a 12-hour light/dark cycle with free access to AIN-76A diet (Research Diets, New Brunswick, NJ) and water. The Institutional Animal Care and Use Committee of the Pennsylvania State University approved all animal experiments.

Isolation of splenic and tumor-in ltrating immune cells
At sacri ce, spleens and tumors were harvested and single cell suspensions of splenocytes and tumorin ltrating immune cells were prepared as previously described (23). Brie y, spleens were mechanically disrupted with a syringe plunger and passed through a 70 µm nylon mesh strainer. Primary tumors were weighed, minced into ne pieces and incubated with 0.03 mg/mL Liberase (Roche, Indianapolis, IN) and 12.5 U/mL DNase I (Thermo Fisher Scienti c, Waltham, MA) for 45 min at 37°C on a rotary sample mixer. Following the digestion, remaining pieces were mechanically disrupted with a syringe plunger and passed through a 70 µm nylon mesh strainer. Following mechanical disruption, both spleen and tumor samples were treated with ACK lysing buffer (Lonza, Basel, Switzerland), washed and resuspended in complete medium. Cell counts and viability were determined via trypan blue (Mediatech) exclusion.

Immune cell assays
Flow cytometric analyses of myeloid and lymphoid cells Single cell suspensions of splenocytes and tumor-in ltrating immune cells were washed with PBS containing 0.1% BSA and incubated with Fc block (BioLegend), followed by staining with Zombie Aqua viability dye (BioLegend). Cells were then stained with uorescent dye-conjugated antibodies against extracellular markers (Additional File 1: Table S2) or the corresponding isotype control antibodies for 30 min at 4°C. CD45 staining was used in each panel to identify total leukocytes. Following antibody incubation, cells were washed twice and xed with BD Cyto x™ xation buffer (BD Biosciences). Additionally, regulatory T cells (Tregs) were assessed using the Mouse Regulatory T Cell Staining Kit (eBioscience) as per manufacturer's instructions. Following extracellular staining for CD4 and CD25, cells were incubated in a Fixation/Permeabilization solution for 30 min, washed twice, and stained with FoxP3 antibody (Additional File 1: Table S2) or the corresponding isotype control antibody for 30 min. After intracellular staining, cells were washed twice and samples were analyzed with the ow cytometer within 12h.

Splenic CD4 + and CD8 + T cells demonstrated the same trend with tumor-in ltrating T cells and was
reduced from D6 to D9 and increased after D9 (Additional File 2: Figure S2). In contrast to T cells, splenic MDSCs were increased from D6 to D9 and reduced after D9. As a result, splenic CD4 + :MDSC and CD8 + :MDSC ratios were reduced from D6 to D9 during the initial tumor growth period and increased after D9 as tumor regression occurred (Additional File 1: Figure S2). The distribution of other myeloid cells and Tregs in the spleen and the tumor are shown in Additional File 2: Figure S3 and Additional File 2: Figure  S4 (Table 1). of IFNγ + cells within CD8 + T cells (Fig. 4B) was unchanged over time, and the percentage of IFNγ + cells within CD49b + NK cells (Fig. 4C) (one-way ANOVA, F(4, 20) = 17.83, p < 0.001) was increased from D9 to D12 and reduced from D15 to D18. The percentage of IL-2 + cells within CD4 + T cells (Fig. 4D) (one-way ANOVA, F(4, 20) = 37.97, p < 0.001) was increased from D6 to D12 and reduced from D12 to D18, the percentage of IL-2 + cells within CD8 + T cells (Fig. 4E) (one-way ANOVA, F(4, 20) = 43.71, p < 0.001) was increased from D9 to D12 and reduced from D15 to D18, and the percentage of IL-2 + cells within CD49b + NK cells (Fig. 4F)  Among splenic effector cells, the percentage of IFNγ + cells and IL-2 + cells within CD4 + T cells, CD8 + T cells and CD49b + NK cells, respectively, was increased from D6 to D15, followed by a reduction at D18 (Additional File 2: Figure S5). The percentage of TNFα + cells within CD4 + and CD8 + T cells was increased from D6 to D9, and the percentage of perforin + cells within CD8 + T cells and CD49b + NK cells was increased at D12-15 compared to D6 (Additional File 2: Figure S5).

4T1.2-HER2 tumor regression occurred concurrently with HER2-speci c IFNγ production
To further characterize antigen-speci c T cell response during the initial tumor growth and regression, we assessed IFNγ secretion by tumor-in ltrating ( Table 2) and splenic (Additional File 1: Table S4) immune cells following ex vivo stimulation with an H-2K d -restricted HER2 peptide, a control HA peptide, anti-CD3/anti-CD28 antibodies, or without stimulus, respectively. Differences between HER2 and HA peptidestimulated groups at each time point were further assessed using paired t test or Wilcoxon test (a twoway ANOVA was not applicable due to non-normal distribution of the data) ( Fig. 5 and Additional File 2: Figure S6). IFNγ secretion by tumor-in ltrating immune cells in HER2 group was higher than HA group at D12 (paired t test, p = 0.030) and D15 (Wilcoxon test, p = 0.063) (Fig. 5). IFNγ secretion by splenic immune cells in HER2 group was higher than HA group at D12 (Wilcoxon test, p = 0.063) (Additional File 2: Figure  S6).   Figure S8). Further, splenic effector to immunosuppressive cell ratios were signi cantly higher in mice that rejected the tumor compared to mice that demonstrated the second phase of tumor growth (Additional File 2: Figure S9).

Discussion
Preclinical cancer models that closely mimic human diseases are valuable tools to study cancer immune responses and develop immunotherapeutic strategies. In the current study, we comprehensively characterized host immune responses in the orthotopic 4T1. In both the spleen and the TME, the percentage of CD4 + and CD8 + T cells was inversely correlated with tumor volume and demonstrated a reduction during the initial tumor growth followed by an increase during tumor regression. However, a stepwise increase in the percentage of splenic and tumor-in ltrating dendritic cells (CD11c + /I-Ad + ) was observed throughout the initial tumor growth and regression, suggesting that the immune system may be recognizing and responding to the 4T1.2-HER2 tumor soon after tumor inoculation. Moreover, there was a stepwise increase in the percentage of IFNγ + , IL-2 + and perforin + T cells and NK cells throughout the initial tumor growth and regression. An increase in splenic TNFα + T cells was also observed during the initial tumor growth. IFNγ production and cytotoxicity are the two most critical anti-tumor effector functions (2,3,32). IL-2 is produced by activated T cells and NK cells and promotes the activation, differentiation and cytotoxicity of effector cells (33,34). TNFα is produced by both myeloid and lymphoid cells with pleiotropic effects, and T cell-derived TNFα has been shown to contribute to anti-tumor immunity (35)(36)(37)(38). Therefore, the observed increase in effector cells expressing these molecules suggest that an anti-tumor immune response may be elicited upon tumor inoculation and mount up throughout tumor growth and regression. Further, ex vivo stimulation with an H-2K drestricted HER2 peptide induced HER2-speci c IFNγ secretion, which peaked during the tumor regression phase at D12-15. When adjusting for cell number, the greatest HER2-speci c IFNγ response was still observed at D12-15. Together, these data suggest that 4T1.2-HER2 tumor regression occurred concurrently with an increase in both the number and functional capacity of effector cells.
During an immune response to infection or cancer, activated T cells can further differentiate into memory cells to provide long-term protection. Memory T cells are categorized into two subsets, central memory (T CM , CD44 + /CD62L + /CCR7 + ) and effector memory (T EM , CD44 + /CD62L − /CCR7 − ) cells, with distinct localization patterns and functional capacity (39)(40)(41)(42). Both T CM and T EM cells play important roles in host immunity, and T CM cells have been shown to confer greater anti-tumor immunity compared to T EM cells when adoptively transferred into hosts (43)(44)(45)(46). In the current study, we observed an increase in splenic and tumor-in ltrating CD4 + and CD8 + T CM cells during tumor regression compared to the initial tumor growth period. This suggests that the anti-tumor immune response occurred concurrently with the formation of immunological memory, which may provide further protection. In human breast cancer, HER2 + and TNBC subtypes are characterized by higher levels of T cell in ltration compared to HR + subtypes (9, 10), and T cell in ltration is negatively correlated with tumor recurrence and mortality in HER2 + and TNBC (11)(12)(13)(14). In contrast to T cells, circulating MDSC levels in breast cancer patients are positively correlated with clinical stage, metastatic tumor burden and treatment with chemotherapy (47)(48)(49). Few clinical studies have assessed MDSC in ltration into the TME. One study reports a signi cant expansion of MDSCs within breast tumors compared to normal breast tissue (50).
Furthermore, MDSC in ltration is greater in the breast cancer tissue of TNBC patients compared to HR + breast cancer patients (51). Consistent with the observations in TNBC patients, the 4T1.2-HER2 model of TNBC induced signi cant T cell in ltration, and tumor progression and regression were associated with changes in the in ltration of effector and immunosuppressive cells into the TME. Therefore, the orthotopic 4T1.2-HER2 model presents a novel, clinically relevant model of TNBC to assess antigenspeci c immune responses.

Conclusions
Results from the current study suggest that the integration of a surrogate tumor antigen, human HER2, into the clinically relevant, yet poorly immunogenic 4T1.2 metastatic breast cancer model enhanced its immunogenicity and induced antigen-speci c immune responses. In contrast to the rapid, continuous tumor growth of the parental 4T1.2 model, 4T1.2-HER2 tumor growth demonstrated a spontaneous regression period that is dependent on the adaptive immune system. Tumor regression occurred concurrently with an increase in the ratio of effector to immunosuppressive cells, the capacity of effector cells to produce cytokine and cytotoxic molecules, and importantly, HER2-speci c immune response as indicated by IFNγ production. Therefore, the orthotopic 4T1.2-HER2 model could be a powerful tool to study the modulation of antigen-speci c immune responses to chemotherapeutic, immunotherapeutic or novel therapeutic interventions to ultimately improve clinical outcomes in breast cancer patients.

Declarations
Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.