Maitake Beta-Glucan Enhances the Therapeutic Effect of Trastuzumab via Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity

Abstract

Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake β-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.

INTRODUCTION

Approximately 15–20% of breast cancers (BCs) overexpress HER2, a transmembrane protein in the ErbB family, which is associated with a more aggressive phenotype and worse prognosis.¹⁾ In 1998, the approval of a humanized monoclonal antibody (mAb), trastuzumab (Herceptin^®), which targets the extracellular domain of HER2, markedly advanced the clinical management of HER2⁺ BC patients.^2–4) In combination with chemotherapy, trastuzumab treatment yields favorable results, but disease progression and relapse still occur in advanced metastatic BC due to the development of resistance to trastuzumab.⁵⁾ Thus, new combination therapies are required to enhance the clinical benefits of trastuzumab further.

Trastuzumab inhibits the growth of tumor cells through multiple mechanisms, including blockade of HER2 signaling and downregulation of the HER2 receptor. In addition, trastuzumab causes antibody-dependent cellular cytotoxicity (ADCC) by binding to the Fcγ receptor (FCGR) on the surface of natural killer (NK) cells and macrophages.^6,7) A limitation of combination therapy using trastuzumab and chemotherapeutic agents, such as taxanes, is that the latter may reduce the antitumor immune response induced by trastuzumab via myelosuppression as a side effect.

Antitumor mAbs, including trastuzumab, activate the classical complement pathway by binding to target cells via C1q, thereby triggering a cascade of enzymatic cleavage reactions involving C3 convertases.^8,9) C3 is cleaved to form C3b, deposited on the target cell membrane as an opsonin. The binding of C3b to target cells leads to complement-dependent cellular cytotoxicity (CDCC) by neutrophils and macrophages via complement receptors (CR1 and CR3). In the complement cascade, membrane-bound C3b interacts with C3 convertase to form C5 convertase, which cleaves C5 and activates the terminal pathway, causing the deposition of the membrane attack complex (MAC), which directly lyses the target cells (complement-dependent cytotoxicity; CDC).¹⁰⁾

Various polysaccharides in plants, fungi, mushrooms, and seaweeds have been reported to have immunostimulatory properties.^11–14) These naturally occurring immunostimulants have been used as adjuvants in other cancer therapies, such as chemotherapy, to prevent the repression of host defenses against infection and enhance overall therapeutic efficacy. We previously demonstrated that β-glucan (named MD-Fraction) extracted from an edible mushroom maitake (Grifola frondosa) exhibits antitumor activity by activating the host immune system.¹⁵⁾ We reported that MD-Fraction enhances cytokine production and antigen-presenting function of macrophages and dendritic cells via the β-glucan receptor, dectin-1,^15,16) shows remarkable antimetastatic activity via activation of NK cells,¹⁷⁾ and induces granulopoiesis via granulocyte colony stimulating factor (G-CSF) production.¹⁸⁾

Expanding on our previous findings, we evaluated the potential use of MD-Fraction to enhance the antitumor effects of trastuzumab using xenograft mouse models. We determined the effects of MD-Fraction on trastuzumab-mediated ADCC. We also examined the effect of MD-Fraction on cytotoxicity via the complement pathway.

MATERIALS AND METHODS

Preparation of MD-Fraction from Maitake Mushroom

MD-Fraction were prepared from maitake fruiting bodies (Yukiguni Maitake, Niigata, Japan) as previously described.¹⁹⁾ Briefly, a hot-water extract of dried maitake powder was subjected to ethanol precipitation, followed by acid precipitation, alkaline extraction, and protein removal using the Sevag method. The water-soluble MD-Fraction showed a single peak corresponding to a molecular weight of 1200–2000 kDa in HPLC. Assessment of MD-Fraction using Endospecy ES-24S set (Seikagaku Biobusiness, Tokyo, Japan) did not reveal any endotoxin contamination.

Cell Lines and Cell Culture

The human HER2⁺ BC cell line, MDA-MB-453, was purchased from RIKEN BRC (Ibaraki, Japan) and maintained in Leibovitz’s L-15 medium (Gibco, Thermo Fisher Scientific, Grand Island, NY, U.S.A.), supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) and antibiotics (penicillin/streptomycin, P/S; Nacalai Tesque Inc., Kyoto, Japan). The human HER2⁺ BC cell line, BT-474, was purchased from ATCC (ATCC No. HTB-20) and maintained in RPMI Medium (Nacalai Tesque Inc.), supplemented with 10% FBS and P/S. The human HER2⁻ BC cell line, MCF-7, was purchased from RIKEN BRC and maintained in α-MEM (Nacalai Tesque Inc.), supplemented with 10% FBS and P/S.

In Vitro Inhibition of BC Growth

Human BC cells (BT-474, MDA-MB-453, and MCF-7; 1 × 10⁶ cells/mL) were stimulated with trastuzumab (Herceptin^®; Chugai Pharmaceutical, Tokyo, Japan) alone, MD-Fraction alone, or a combination of trastuzumab and MD-Fraction for 24 h and the percentage of viable cells was examined with the WST-8 reagent using the Cell Count Reagent SF (Nacalai Tesque Inc.).

In Vivo Tumor Model

Female athymic BALB/c nu/nu and DBA/2 mice (5–8 weeks) were purchased from CLEA Japan (Tokyo, Japan). To prepare BC-bearing animals, a suspension of 1 × 10⁷ BT-474 or MDA-MB-453 cells/50 µL was injected subcutaneously into the left flank of BALB/c nu/nu mice together with 50 µL of Matrigel^® (BD Biosciences, San Jose, CA, U.S.A.). A 0.05 mL estrogen suspension (Pellanin Depot^®, Mochida Pharmaceutical, Tokyo, Japan) was injected into the thigh muscle of BT-474 BC-bearing mice once a week. MD-Fraction (8 mg/kg) was administered intraperitoneally thrice a week, starting the day after tumor inoculation. Trastuzumab (1 mg/kg) was administered intraperitoneally twice a week, beginning two weeks after tumor inoculation. Tumor length and width were measured using calipers every 2–3 d. The tumor volume was calculated using the following equation: tumor volume (cm³) = (tumor length × tumor width²)/2.

All animal experiments complied with the ARRIVE guidelines and were performed following the National Research Council Guide for the Care and Use of Laboratory Animals and the Guidelines of the Animal Care and Use Committee of Kobe Pharmaceutical University (Permit Number: 2014-026). Mice were sacrificed when the longest tumor diameter reached 2 cm. At the end of the experiments, the mice were anesthetized with isoflurane and sacrificed by cervical dislocation. All efforts were made to minimize animal suffering.

Real-Time Quantitative (q) PCR

Real-time qPCR was used to examine mRNA expression levels. Total RNA was purified from cells or tissues using Sepasol RNA I Super (Nacalai Tesque Inc.), and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). qPCR was performed using Thunderbird SYBR qPCR mix (Toyobo) and primers for 18s, Il12a, Tnf and Il1b, as described previously.^16,20) Other primer sequences were as follows: Ifng, forward primer 5′-TGAGCTCATTGAATGCTTGG-3′; Ifng reverse primer, 5′-ACAGCAAGGCGAAAAAGGAT-3′; Ccl2 forward primer, 5′-TTAAAAACCTGGATCGGAACCAA-3′; Ccl2 reverse primer, 5′-GCATTAGCTTCAGATTTACGGGT-3′; erbb2 forward primer, 5′-GATCATCATGGAGCTGGC-3′; erbb2 reverse primer, 5′-GTCGCAACTTCATGTCGGTA-3′.

Flow Cytometry

Single-cell suspensions of the spleen were prepared by passing the tissue homogenate through a 70 µm nylon strainer (BD Biosciences) and were used for further experiments. Splenocytes were stained with the following mAbs and isotype-matched control antibodies for 20 min at 4 °C; anti-mouse CD49b fluorescein isothiocyanate (FITC) (clone DX5), anti-mouse Ly6G PE (clone 1A8), and anti-mouse CD69 PE (clone H1.2F3) from BD Pharmingen (San Diego, CA, U.S.A.) and anti-mouse CD11b PE-Cy7 (clone M1/70), and anti-mouse F4/80 FITC (clone BM8) from BioLegend (San Diego, CA, U.S.A.). The stained cells were washed and analyzed using a flow cytometer (FACSCalibur; BD Biosciences). Data were analyzed using FACSDiva (BD Biosciences) and FlowJo software (Tree Star, Ashland, OR, U.S.A.).

Preparation of Immune Cells

Peripheral blood mononuclear cells and splenic NK cells were prepared as described below. BALB/c nu/nu mice were intraperitoneally injected with MD-Fraction (8 mg/kg) or phosphate-buffered saline (PBS) (as a control) for two consecutive days, starting a day after MDA-MB453 transplantation and peripheral blood and spleen were collected. The total lymphocytes were isolated from the peripheral blood using Lympholyte-M solution (Cedarlane Laboratories Ltd., Burlington, Canada). Splenic NK cells were isolated from the splenocytes using anti-CD49b MicroBeads (clone DX5; Miltenyi Biotec, Bergisch Gladbach, Germany) with >85% purity.

As macrophages from DBA/2 mice were previously found to be sensitive to MD-Fraction, a dectin-1 ligand, in vitro macrophage experiments were performed using DBA/2 mice.¹⁶⁾ Resident peritoneal cells were harvested using peritoneal lavage of DBA/2 mice with PBS (−). Adherent cells were enriched by adherence to plastic in RPMI-1640 supplemented with 10% FBS and P/S for 2 h and used as macrophages. Macrophages (1 × 10⁶ cells/mL) were then stimulated with MD-Fraction (50 µg/mL) for 24 h.

To prepare splenic neutrophils, MD-Fraction (8 mg/kg) was administered intraperitoneally to BALB/c mice for three consecutive days, and neutrophils were separated from splenocytes using Anti-Ly-6G MicroBeads (Miltenyi Biotec) with >85% purity.

Cytotoxicity Assay

To fluorescently label target tumor cells, they (1 × 10⁶ cells) were suspended in 1 mL of culture medium; 0.1% calcein-acetoxymethylester (Calcein-AM; Dojindo Laboratories, Kumamoto, Japan) was added to it, and the cell suspension was incubated at 37 °C for 45 min. Thereafter, the cells were washed twice with PBS and resuspended in the culture medium at 1 × 10⁵ cells/mL. The tests were performed as follows: 50 µL each of effector immune cells and Calcein-AM labeled target cells (MDA-MB-453 or BT-474 cells) were added to a 96-well U-bottom plate (BD Falcon, Franklin Lakes, NJ, U.S.A.) at ET ratios of 2–60 : 1 and cocultured for 2 h, as previously described.¹⁷⁾ For CDCC assay, Calcein-AM labeled BT-474 cells were pretreated with trastuzumab (1 µg/mL) for 30 min in the presence of 1% native mouse serum or heat-inactivated (HI) mouse serum, and the effector immune cells were subsequently added and cocultured for 2 h. HI mouse serum was obtained by heat treatment of DBA/2 mouse serum at 56 °C for 30 min. After incubation, the fluorescence intensity of the supernatant was measured as Calcein-AM release from the target cells using Fluoromark (Bio-Rad Laboratories, Hercules, CA, U.S.A.) at an excitation wavelength of 485 nm and a detection wavelength of 538 nm. Cytotoxicity was calculated using the following equation: cytotoxicity (%) = [(test release − spontaneous release)/(maximum release − spontaneous release)] × 100.²¹⁾ Spontaneous release was defined as the release of Calcein-AM from target cells in the medium only, and maximal release was defined as the release of Calcein-AM from target cells lysed in 3% Triton X-100-added medium.

Activation and Deposition of Complement on Tumor Cells, and CDC Assay

BT-474 cells, fluorescently labeled with Calcein-AM, were incubated with 20% mouse serum or HI mouse serum and trastuzumab (1 µg/mL) for 30 min and then MD-Fraction (0, 10, 100 µg/mL) was added. After 1 h of incubation, C3b deposition on BT-474 cells was measured by flow cytometry with FITC-labeled anti-human/mouse C3/C3b/iC3b (clone: 6C9, Cedarlane Laboratories Ltd., Burlington, Canada). The concentration of C3a in the culture supernatant was measured using Mouse C3a enzyme-linked immunosorbent assay (ELISA) Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). After 24 h of incubation, CDC was examined by measuring the amount of fluorescence in the culture supernatant.

Statistical Analysis

Data were analyzed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, U.S.A.). Data are expressed as means ± standard deviation (S.D.; in vitro experiments) or means ± standard error of the mean (S.E.M.; ex vivo and in vivo studies). One-way ANOVA with Tukey’s post-hoc test was used to analyze multiple groups and Student’s t-test was used to compare two groups. Tumor growth data were analyzed using two-way ANOVA. Differences were considered statistically significant at p < 0.05.

RESULTS

MD-Fraction Does Not Directly Inhibit the Growth of HER2⁺ BC Cells and Does Not Affect the Antiproliferative Effect of Trastuzumab

Among the human BC cells used in this study, the expression of erbb2 (HER2 mRNA) was the highest in BT-474 cells, moderate in MDA-MB453 cells, and lowest in MCF-7 cells (Fig. 1A), as reported previously.²²⁾ To examine the effect of MD-Fraction on the growth of these BC cells, the cells were incubated with MD-Fraction (0–50 µg/mL) for 24 h. MD-Fraction did not directly inhibit the cell growth (Fig. 1B). Trastuzumab inhibited the growth of BC cells in a HER2 expression level-dependent manner, and its growth-inhibitory effect was not affected by co-treatment of cells with MD-Fraction (Fig. 1C). These results support previous findings that MD-Fraction does not act directly on tumor cells, but inhibits tumor growth via the immune system.^15,16)

Fig. 1. MD-Fraction Does Not Affect the Antiproliferative Effect of Trastuzumab in HER2⁺ BC Cells

(A) Expression of HER2 mRNA in BT-474, MDA-MB-453, and MCF-7 cells was analyzed using RT-PCR. Relative expression levels normalized to the expression levels in MCF-7 are shown. (B) Human BC cells (1 × 10⁶ cells/mL) were stimulated with MD-Fraction for 24 h. (C) Human BC cells (1 × 10⁶ cells/mL) were stimulated with trastuzumab or trastuzumab plus MD-Fraction (100 µg/mL) for 24 h. Cell viability was determined using the WST-8 reagent (B, C). Data are expressed as means of values obtained in 3–5 replicate experiments (bars, standard deviation). BC, breast cancer; MD, MD-Fraction; TRA, trastuzumab.

MD-Fraction Enhances the Antitumor Activity of Trastuzumab in Human BC Xenograft Model Mice

Next, we examined whether MD-Fraction enhanced the therapeutic effect of trastuzumab in vivo using xenograft models in which human HER2-positive BC cells were subcutaneously transplanted into BALB/c nude mice. As shown in Fig. 2A, 27 d after transplantation of MDA-MB-453 cells, tumor growth was inhibited by 44.5% in MD-Fraction alone group (p = 0.07 vs. PBS). In contrast, it was strongly inhibited by 87.4% (p = 0.0001 vs. PBS) in trastuzumab alone group. Treatment with the combination MD-Fraction and trastuzumab inhibited the tumor growth most strongly by 94.6% (p < 0.0001 vs. PBS) in this xenograft model, but this effect was not significantly different from each treatment alone (p > 0.05). As shown in Fig. 2B, 49 d after transplantation of BT-474 cells, tumor growth was inhibited by 46.1% (p < 0.01 vs. PBS) in MD-Fraction alone group and by 61.9% (p < 0.0001 vs. PBS) in trastuzumab alone group. Treatment with the combination of MD-Fraction and trastuzumab showed the highest tumor growth inhibition of 95.4% (p < 0.0001 vs. PBS) in this xenograft model, which was significantly more effective than each treatment alone (p < 0.05), indicating that this combination therapy is effective in HER2⁺ BCs.

Fig. 2. MD-Fraction Enhances Trastuzumab Efficacy in HER2⁺ BC Xenograft Models

BALB/c nude mice implanted with MDA-MB-453 (A) or BT-474 (B) cells were treated with PBS or MD-Fraction (8 mg/kg) three times a week, starting a day after implantation and trastuzumab (1 mg/kg) twice a week, starting two weeks after implantation. Tumor volumes were measured and are presented as mean + standard error of mean (n = 4 for MDA-MB-453, n = 4–5 for BT-474, * p < 0.05, vs. singe-agent groups). BC, breast cancer; MD, MD-Fraction; PBS, phosphate-buffered saline; TRA, trastuzumab.

MD-Fraction Enhances Trastuzumab-Mediated ADCC by Activating NK Cells

Athymic nude mice lack peripheral T cells, but retain NK cells and macrophages, which exhibit higher cytotoxic activity than euthymic mice.^23,24) Therefore, we investigated the possible contribution of immune cells to the enhanced antitumor effect of MD-Fraction on trastuzumab in xenograft models. Administration of MD-Fraction to tumor-free nude mice significantly increased the spleen weight (Fig. 3A). In addition, MD-Fraction significantly increased the number of NK cells, macrophages, and neutrophils in the spleen (Fig. 3B). As shown in Fig. 3C, MD-Fraction increased the expression of the activation marker, CD69, on NK cells, which are the main effector cells in ADCC. These results suggest that MD-Fraction can activate NK cells and enhance trastuzumab-induced ADCC. To determine whether MD-Fraction promotes trastuzumab-induced ADCC, we isolated peripheral blood lymphocytes and splenic NK cells from MDA-MB-453-bearing mice and measured their ex vivo cytotoxic activity against MDA-MB-453 cells in the presence of trastuzumab. As shown in Fig. 3D, the percentage lysis of MDA-MB-453 cells was significantly higher for peripheral blood lymphocytes from mice treated with MD-Fraction than for those from PBS-treated control mice. Furthermore, splenic NK cells isolated from MD-Fraction-treated mice showed significantly higher cytotoxic activity against MD-MBA-453 cells than those isolated from PBS-treated control mice (Fig. 3E). The expression of different mRNAs in the spleens of MDA-MB-453-bearing mice treated with MD-Fraction was quantified using RT-PCR (Fig. 3F). Treatment with MD-Fraction significantly increased the expression of Ifng, Ccl2, and Il1b. Interferon-γ (IFN-γ) is mainly produced by activated NK cells, whereas CCL2 and interleukin (IL)-1β are produced by activated macrophages. These results suggest that treatment with the combination of trastuzumab and MD-Fraction enhances trastuzumab-mediated ADCC activity, thereby improving the in vivo antitumor efficacy.

Fig. 3. MD-Fraction Significantly Augments Trastuzumab-Mediated ADCC ex Vivo

(A–C) MD-fraction (8 mg/kg) was administered intraperitoneally to BALB/c nude mice thrice a week, and spleen weight (A) was measured on day 7. (B) The numbers of NK cells (CD49b⁺), macrophages (F4/80⁺), and neutrophils (Ly-6G⁺) in the spleen were measured by flow cytometry. (C) The expression of the activation marker CD69 in splenic NK cells is indicated using MFI. (D–F) MDA-MB453-bearing nude mice were treated with MD-Fraction (8 mg/kg) or PBS for two days, and PBMCs (D) or splenic NK cells (E) were harvested on day 3 and used as effector cells. As target cells, Calcein-AM-labeled MDA-MB-453 cells were pretreated with trastuzumab (1 µg/mL) for 30 min and subsequently cocultured with effector cells for 2 h at the ratio indicated. ADCC lysis was assessed by measuring the release of Calcein-AM. (F) Expression of mRNAs in the spleen was measured by RT-PCR. Data are shown as mean ± standard error of the mean (n = 4, * p < 0.05; ** p < 0.01). ADCC, antibody-dependent cellular cytotoxicity; MD, MD-Fraction; MFI, mean fluorescence intensity; NK cells, natural killer cells; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; TRA, trastuzumab.

MD-Fraction-Treated Macrophages and Neutrophils Enhance Trastuzumab-Mediated Cytotoxicity the Presence of Native Serum but Not HI Serum

The fact that MD-Fraction increased the number of macrophages and neutrophils and the expression of inflammation-related molecules, such as Ccl2 and Il1b (Figs. 3B, F) suggested that MD-Fraction may also enhance the cytotoxic effect of trastuzumab via macrophages and neutrophils. MD-Fraction has been reported to directly activate peritoneal macrophages in vitro.¹⁶⁾ However, macrophages stimulated with MD-Fraction did not enhance trastuzumab-mediated cytotoxicity against BT-474 cells in the presence of HI serum (Fig. 4A). Interestingly, in the presence of native serum with complement activity, MD-Fraction-stimulated macrophages significantly enhanced trastuzumab-induced cytotoxic activity (Fig. 4A). Neutrophils isolated from the spleen showed little tumor cell cytotoxicity in the presence of HI serum but showed high cytotoxicity in the presence of native serum (Fig. 4B). Neutrophils isolated from the spleen of mice treated with MD-Fraction increased trastuzumab-induced cytotoxicity in the presence of native serum, although not significantly, compared with neutrophils from PBS-treated mice (Fig. 4B). The Fc region of antibodies is known to activate the classical complement pathway, causing CDCC.⁹⁾ These results indicate that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced cytotoxic activity against HER2⁺ BC cells, suggesting that a heat-sensitive component in the serum, viz., complement, may be involved in this process.

Fig. 4. MD-Fraction-Treated Macrophages and Neutrophils Enhance Trastuzumab-Mediated Lysis of HER2⁺ BC Cells in the Presence of Native Serum

(A) Peritoneal macrophages were stimulated in the presence or absence of MD-Fraction (50 µg/mL) for 24 h. Calcein-AM-labeled BT-474 cells were pretreated with trastuzumab (1 µg/mL) for 30 min in the presence of 1% native serum or HI serum; macrophages (E/T = 20) were then added and the cells were cocultured for 24 h. Lysis of BT-474 cells was measured using Calcein-AM release. (B) BALB/c nude mice were treated with MD-Fraction (8 mg/kg) or PBS for 2 d, and splenic neutrophils were harvested on day 3 and used as effector cells. Calcein-AM-labeled BT-474 cells were pretreated with trastuzumab (1 µg/mL) for 30 min in the presence of 1% native serum or HI serum; neutrophils (E/T = 20) were then added and the cells were cocultured for 2 h. Lysis of BT-474 cells was measured using Calcein-AM release. Data are shown as mean ± standard error of the mean. Statistical differences versus * without MD-Fraction and # HI vs. native serum are indicated (n = 5, * p < 0.05, #p < 0.05, ##p < 0.01, ###p < 0.001). BC, breast cancer; HI, heat-inactivated; MD, MD-Fraction; PBS, phosphate-buffered saline; TRA, trastuzumab.

MD-Fraction Increases Trastuzumab-Associated CDC

As described in “MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-mediated cytotoxicity the presence of native serum but not HI serum,” our results indicate that the complement pathway may be involved in the cytotoxic effects of MD-Fraction-treated macrophages and neutrophils (Fig. 4). C3 fragments, C3b, and its inactive form, C3b (iC3b), are important opsonins that enhance immune effector cell function by binding to complement receptors (CRs) on macrophages and neutrophils.²⁵⁾ Therefore, to investigate whether MD-Fraction induces the deposition of C3b/iC3b on the surface of target tumor cells, BT-474 cells were treated with MD-Fraction in the presence of trastuzumab and native serum, and the deposition of C3b/iC3b on BT-474 cells was detected using a FITC-labeled specific antibody. As shown in Fig. 5A, the deposition of C3b/iC3b on BT-474 cells increased when native serum was added compared to when HI serum was added but was not affected by MD-Fraction. However, the C3 fragment, C3a, increased in a dose-dependent manner with the addition of MD-Fraction (Fig. 5B), suggesting that MD-Fraction could activate the complement pathway. To examine CDC, the lysis of BT-474 cells was measured when MD-Fraction was added in the presence of native serum and trastuzumab. MD-Fraction enhanced trastuzumab-induced CDC dose-dependently (Fig. 5C).

Fig. 5. MD-Fraction Enhances Trastuzumab-Induced CDC

(A) BT-474 cells (1 × 10⁶ cells/mL) were added to 20% mouse native serum or HI serum, and then trastuzumab was added to both; the cells were pretreated for 30 min. MD-Fraction (0, 10, 100 µg/mL) was added to the cell suspension, followed by a 1-h incubation. The deposition of C3b on BT-474 was then measured using flow cytometry with FITC-labeled anti-C3b antibody (n = 4). (B) The concentration of C3a in the culture supernatant was determined using ELISA (n = 6). (C) BT-474 cells (1 × 10⁵ cells/mL) fluorescently labeled with Calcein-AM were incubated with 20% native or HI serum and trastuzumab (1 µg/mL) for 30 min. MD-Fraction was then added, followed by further incubation for 24 h. Lysis of tumor cells was measured using Calcein-AM release (n = 5). Data are shown as mean ± standard error of the mean (** p < 0.01, *** p < 0.001). CDC, complement-dependent cytotoxicity; ELISA, enzyme-linked immunosorbent assay; FITC, isothiocyanate; HI, heat-inactivated; MD, MD-Fraction; MFI, mean fluorescence intensity; TRA, trastuzumab.

DISCUSSION

Trastuzumab, a humanized monoclonal antibody, significantly improves the management and outcomes of patients with HER2-overexpressing BC. Despite the clinical benefits of trastuzumab, some patients develop primary resistance, and even those who achieve an initial response often develop resistance.^26,27) Therefore, substantial research has been conducted to develop novel therapeutic approaches to augment the therapeutic effects of trastuzumab. This study shows that MD-Fraction, maitake β-glucan, has the potential to enhance the antitumor activity of trastuzumab when used in combination with this monoclonal antibody. MD-Fraction did not directly inhibit the survival of HER2⁺ BC cells, either alone or in the presence of trastuzumab, but did enhance the cytotoxic effect of trastuzumab via the immune system.

ADCC is one of the important mechanisms underlying the action of trastuzumab. It occurs when antibodies bind to tumor cells and the Fcγ region of the antibody is recognized by immune cells expressing the FcγR, such as NK cells and macrophages.^6,9) Human FcγRs do not bind to or have low binding affinity for mouse immunoglobulin G (IgG) subclasses, whereas mouse FcγRs bind to human IgG subclasses efficiently.²⁸⁾ Therefore, xenograft models and ex vivo experiments using mouse immune cells could be used to evaluate ADCC responses to trastuzumab. Administration of MD-Fraction increased the number of NK cells, macrophages, and neutrophils in the spleen of nude mice (Fig. 3B). Previous studies have shown that MD-Fraction activates NK cells and increases their cytotoxicity against tumor cells.¹⁷⁾ Peripheral blood lymphocytes and splenic NK cells isolated from nude mice treated with MD-Fraction enhanced trastuzumab-induced ADCC ex vivo (Figs. 3D, E), indicating that MD-Fraction enhances NK cell-dependent ADCC induced by trastuzumab.

Trastuzumab is an IgG1 antibody that activates the classical complement pathway.^29,30) Upon activation of the complement system, (1) anaphylatoxins (C3a and C5a) mobilize immune cells, (2) MAC (C5b-9) formed on tumor cells perforates the cell membrane and lyses the cells (CDC), and (3) tumor cells are opsonized (C3b and its inactive form iC3b), which are then killed by immune cells (CDCC).³¹⁾ Treatment of HER2⁺ BCs with MD-Fraction in the presence of trastuzumab and native serum increased the release of C3a and lysis of tumor cells in a dose-dependent manner (Figs. 5B, C). These results suggested that MD-Fraction enhanced trastuzumab-induced CDC by activating the complement pathway. Zymosan, a crude particulate β-glucan derived from the cell wall of Saccharomyces cerevisiae, is a well-known stimulator of the antibody-independent alternative complement pathway.^32,33) The mechanism of complement activation by MD-Fraction needs to be clarified in future studies. On the contrary, MD-Fraction did not affect C3b/iC3b opsonization on tumor cells by trastuzumab (Fig. 5A). Because yeast β-glucans have been reported to bind directly to C3b/iC3b,³⁴⁾ it is possible that the C3b/iC3b released from C3 is partially bound to MD-Fraction.

C3b and iC3b opsonized on tumor cells acts as ligands for CRs, such as CR1 (CD35), CR3 (CD11b/CD18), and CRIg (CR of the immunoglobulin superfamily), which are expressed on effector cells, such as macrophages and neutrophils.⁹⁾ Because CR3 needs to be preactivated to bind to opsonized tumor cells, CR3 is usually considered to contribute little to mAb therapy.³⁵⁾ Yeast-derived β-glucan induces an antitumor immune response by specifically activating CR3 on macrophages and neutrophils.^34,36) The binding of yeast β-glucan to the CD11b subunit of CR3 activates the I-domain of the CD18 subunit of CR3, allowing it to bind to iC3b-opsonized tumor cells. In response to iC3b-opsonized tumor cells, macrophages and neutrophils primed with yeast β-glucan produce cytokines, such as tumor necrosis factor (TNF)-α, IFN-γ, IFN-α, and IL-6, which are associated with the destruction of tumor cells.³⁷⁾ MD-Fraction-treated macrophages and neutrophils did not enhance the cytotoxicity of trastuzumab in the presence of HI serum, but they did in the presence of native serum (Fig. 4), indicating that macrophages and neutrophils treated with MD-Fraction lysed cancer cells in a complement-dependent manner. MD-Fraction has previously been shown to induce the production of inflammatory cytokines via dectin-1 in macrophages and partially via CD11b to induce cell proliferation.¹⁶⁾ It is possible that MD-Fraction causes CDCC by binding to CD11b on macrophages and neutrophils.

Combining antitumor mAbs, such as trastuzumab, with immunotherapy, such as cytokine or immune checkpoint therapy, is a new antitumor strategy.³⁸⁾ Our findings supported this hypothesis. Combination with MD-Fraction could be effective in enhancing the therapeutic effect of trastuzumab because it induces the activation of NK cells, macrophages, neutrophils, and complement pathways, which are important in the mechanism underlying the anticancer effect of trastuzumab. Furthermore, MD-Fraction, derived from edible mushrooms, has shown no side effects in mouse experiments and human clinical trials^39–41) and may be a safe addition to trastuzumab-based cancer treatments.

Our study demonstrates that the combination of trastuzumab with MD-Fraction has greater antitumor efficacy than trastuzumab alone by enhancing ADCC, CDCC, and CDC against HER2⁺ BC, both ex vivo and in vivo. This study provides evidence for the therapeutic efficacy of trastuzumab and MD-Fraction in the treating HER2⁺ BCs.

Acknowledgments

We thank A. Kawasaki, Y. Makino, and Y. Watanabe (Department of Microbial Chemistry, Kobe Pharmaceutical University, Japan) for their technical assistance.

Funding

This work was supported by Yukiguni Maitake Co., Ltd. and by Scholarship Fund for Young Researchers from The Promotion and Mutual Aid Corporation for Private Schools of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

Data will be made available on request.

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