Doxorubicin, Taxol, etoposide and cisplatin were acquired from Sigma Aldrich; Merck KGaA. Each chemotherapeutic agent was used in incrementally increased concentrations (from 0 to 3 mg/ml) to 1 mg/ml of the LCS102 preparation (described in detail below) for 30 min each. RPMI-1640, L-glutamine, FBS, trypsin and PBS were purchased from Biological Industries. Red blood cell lysis buffer, Alexa Fluor 488-conjugated mouse anti-human CD56 IgG1κ (cat. no. 318312; CD56-AF), PE-conjugated mouse anti-human CD69 IgG1κ (cat. no. 310906; CD69-PE), PerCP-conjugated mouse anti-human CD3 IgG1κ (cat. no. 344814 CD3-PerCP), PE-conjugated mouse IgG1κ MOPC-21 isotype control antibody, Alexa Fluor 488-conjugated mouse IgG1κ MOPC-21 isotype control antibody and PerCP-conjugated mouse IgG1κ MOPC-21 isotype control antibody were all purchased from BioLegend, Inc. IL-2 was from purchased PeproTech, Inc.

The present study was approved by the Sheba Medical Center Institutional Review Board (Ramat Gan, Israel). For the present study, 20 volunteers (12 male, 8 female) were asked to donate blood, 19 of these were healthy donors with no chronic medical illness or medication use, including drugs with a potential to alter the immune response; and 1 of the female patients had a carcinoma of the breast and was undergoing chemotherapy. All volunteers provided written informed consent, and 5 ml of blood was drawn from each donor and collected in heparin-treated tubes (Greiner Bio-One, GmbH). Each tube was inverted several times and then treated as described below.

A549 (lung carcinoma), MCF7 (breast adenocarcinoma), PANC-1 (pancreatic epithelioid carcinoma), U-2 OS (osteosarcoma) and T24 (bladder transitional cell carcinoma) were purchased from American Type Culture Collection, and were authenticated using STR profiling. All cells were cultured in RPMI-1640 with 10% FBS, 2 mM L-glutamine, 100 µg/ml Pen/Strep (Biological Industries) in a 37˚C humidified incubator with 5% CO₂.

LCS102 is comprised of the following herbal components: Astragalus membranaceus, Poriae cocos, Atractylodes macrocephala, Ligustrum lucidum, Lycium chinense, Ganoderma lucidum and Cordyceps sinensis, extracts of which are manufactured in accordance with good manufacturing practice guidelines, and imported under license (BARA Herbs, Yokneam, Israel) in accordance with the regulations of the Israel Ministry of Health. All batches of the final product were analyzed and certified to be free of contaminants, such as heavy metals, microbial contamination, pesticide residues and mycotoxins. A dry extract powder of the LCS102 and LCS101 formulas (BARA Herbs Ltd) was dissolved in PBS at a concentration of 100 mg/ml, and incubated for 30 min with heating and occasional vortexing. The solution was then centrifuged at 4,300 x g for 5 min, and the supernatant was filtered through a 0.45 µm Millex PVDF filter (Merck KGaA). Solubility was estimated by cryophilization and weighing of the pellet, and was estimated to be ~50%.

Blood samples (100 µl) were plated in U-shaped 96-well plates and treated in triplicate immediately with 0-3 mg/ml of the botanical extracts. After 48 h of incubation at 37˚C in a 5% CO₂ incubator treated aliquots (50 µl) were stained with 3 µl 1:1:1 CD56-AF/CD69-PE/CD3-PerCP or an isotype-matched control antibody mix for 30 min at room temperature, lysed with red blood cell lysis buffer, washed twice with PBS and re-suspended in PBS for subsequent FACS analysis. In all experiments PBS-treated samples were used as a negative control, and an IL-2 (20 U/ml)-treated sample (prepared at the same temperature and for the same duration as the botanical compounds) as the positive control.

The samples were analyzed using a BD FACSCalibur flow cytometer (BD Biosciences), using a three-color protocol with appropriate compensations. The results were analyzed using WinMDI version 2.9 (Purdue University Cytometry Laboratories) using the following methodology: First, a forward scatter (FSC)/ side scatter (SSC) dot plot was used to select neutrophil (R1, blue) and lymphocyte (R2, green) populations (Fig. 1A). Lymphocytes were further resolved using a CD3-PerCP/CD56-AF488 logarithmic dot plot (Fig. 1B) to select CD3^-/CD56⁺ NK cells (R4, purple) population. Finally, each population was analyzed for CD69 expression on a CD69-PE logarithmic histogram plot, with appropriate gating (neutrophils, Fig. 1C; NK cells, Fig. 1D). An activated population marker (M1) was set in accordance with the isotype control staining of each population (data not shown). Due to differences in self-fluorescence, the settings for the activation markers differ between NK cells and neutrophils.

Sulforodamine B, trichloroacetic acid and acetic acid were purchased from Sigma Aldrich; Merck KGaA. A total of 3x10³ cells were plated per/well in a 96-well plate and incubated overnight. Subsequently, cells were treated in triplicates as described above and cultured for 48 h. A SRB viability test was performed as follows: Cells were fixed for 1 h on ice with 10% trichloroacetic acid (v/v in RPMI-1640), washed trice with double distilled water, and dried and stained for 1 h at room temperature with 0.057% sulforodamine B (w/v in 1% acetic acid). After staining, the plates were washed three times with 1% acetic acid and then dried, and 200 µl 10 mM Tris was added to each well to solubilize the SRB. The absorbance was measured at 570 nm using an ELISA reader (Biotek Instruments, Inc.). Each experiment was repeated at least three times.

A total of 10 study groups were assessed, each in triplicate: LCS102, controls, IL-2, AME, AMA, PCAO, LCH, LLU, GLU and CSI. A total of 45 between-group comparisons were performed, using a non-parametric Kruskal-Wallis test, with a post-hoc Dunn's test. The data were analyzed using SPSS version 25 (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

The effects of the LCS102 formula on neutrophil and NK cell activation were examined in all of the blood samples. Samples were treated with 1 mg/ml of LCS102, PBS (negative control) or IL-2 (positive control) for 48 h, stained with a CD3/CD56/CD69 fluorescent antibody mix and then analyzed using FACS. Neutrophil populations were gated on an FSC/SSC dot-plot (Fig. 1A), and analyzed on a CD69-PE histogram (Fig. 1C). NK cells were first gated on an FSC/SSC dot-plot (Fig. 1A) for lymphocytes, and then on a CD3-PerCP/CD56-AF488 dot-plot for CD3^-/CD56⁺ (NK) cells (Fig. 1B), and analyzed on a CD69-PE histogram (Fig. 1D).

Fig. 2 shows the percentage of activated NK/neutrophils above/below the negative control value in each sample, as well as the average value of all samples for each treatment. LCS102 significantly elevated the percent of activated neutrophils and NK cells in the majority of tested blood samples, although the extent of this activation varied among samples.

The effects of LCS102 on cancer cell survival were assessed and compared to with the LCS101 formula on a panel of five human cancer cell lines. As shown in Fig. 3A, LCS102 reduced the proliferation of two cancer cell lines, U-2 OS and MCF7, although it was less effective than the anti-cancer effects of LCS101. In order to address the effects of both formulas on NK and neutrophil activation, blood samples from three donors were treated with increasing (0 to 3 mg/ml) concentrations of the compounds. As shown in Fig. 3B, LCS102 was considerably more effective in the activation of NK and neutrophils compared with LCS101.

Each cell line was exposed to incremental concentrations of cisplatin (cisPL), paclitaxel (Taxol), etoposide (VP-16) and doxorubicin (doxo). The addition of 1 mg/ml of LCS102 to each of the chemotherapy-treated cells did not result in reduced cytotoxic effects for any of the treatments in any of the cancer cell lines assessed. Additionally, in Panc-1 cells, the addition of LCS102 to doxorubicin resulted in a synergistic response, which manifested as increased cell death. This response was also observed with the addition of LCS102 to 1 µM of cisplatin in T-24 cells (Fig. 4).