PSK was donated by Kureha Chemical Industry (Tokyo, Japan) and docetaxel obtained from Sanofi-Aventis USA (Bridgewater, NJ).

CD4 and CD8 primary Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Ki67 was obtained from Dako (Copenhagen, Denmark). The TUNEL staining kit was obtained from R&D Systems (Minneapolis, MN). The ABC Vector staining kit (Vector Labs, Burlingame, CA) was used for fluorescent secondary staining in some cases. Rabbit polyclonal IgG against cleaved caspase-3 (Asp 175) (specific for mouse, rat and human cleaved caspase-3) was obtained from Cell Signaling Technology (Beverley, MA).

Mouse prostate tumor (TRAMP-C2) cells (American Type Culture Collection, Manassas, VA) were injected orthotopically into C57BL/6 mice (Charles River, Wilmington, MA) following a study protocol approved by the University of Minnesota Institutional Animal Care and Use Committee. This procedure was performed under general anesthesia (isoflurane). Tumors were allowed to grow for approximately two weeks. Tumor growth was monitored by palpation of the prostate; established tumors present as hardened masses at the injection site. After this period, mice were randomly assigned to one of four treatment groups: 1) saline control; 2) docetaxel only; 3) PSK only; 4) docetaxel plus PSK. Control mice received saline daily through oral gavage, as well as twice weekly saline intraperitoneal (i.p.) injections. The docetaxel only treatment group received docetaxel (5 mg/kg) injected i.p. twice weekly and saline vehicle control daily by oral gavage. The PSK only treatment group received PSK (300 mg/kg) daily by oral gavage and i.p. injections of saline twice weekly. The docetaxel + PSK treatment group received PSK (300 mg/kg) daily by oral gavage and docetaxel (5 mg/kg) i.p. injections twice weekly. Injections were administered in 100 μl volumes. Mice were weighed every other day. Groups were treated for 11–13 days before mice were sacrificed and specific organs harvested for end-point monitoring. Prostate tumors were excised and weighed, and tumors and spleens were used to determine concentration and activity of different immune cell types. Excised tumors were also analyzed for proliferation and apoptosis. Blood was also collected to determine white blood cell counts and assay hepatic and renal function for safety assessment.

To confirm the presence of tumor, frozen sections of tumor tissue (4 micron) were subjected to hematoxylin and eosin staining. Sections were placed into distilled water and nuclei stained with alum hematoxylin for 4 minutes (min), then rinsed with water. Sections were then treated with 0.3% acid alcohol and rinsed. Slides were then treated with eosin for 2 min, rinsed, dehydrated, mounted and examined for tumor presence.

Unstained serial frozen sections of tumor were cut to 4-micron thickness and placed into phosphate buffered saline (PBS). Sections were exposed to 1–1,000 dilution of Ki67 (Dako) and incubated for 1 hour (h) at 37°C. All slides were incubated with the appropriate biotinylated secondary antibody: goat anti-rabbit IgG (Vector), for 20 min at 37°C. The cells were then counted as described below. Other nearby sections were used for immuno-fluorescent staining to detect the presence of apoptosis using the TUNEL apoptosis assay kit (R&D Systems) according to the manufacturer's protocol. Briefly, slides were treated with proteinase K for 30 min at 37°C and incubated with a terminal deoxynucleotidyl transferase end labeling cocktail (terminal deoxynucleotidyl transferase buffer, biotin-dUTP, and terminal deoxynucleotidyl transferase at a ratio of 90:5:5) for 120 min at 37°C. This was followed by overlaying an avidin-FITC (green) solution (50 μl) and incubating in the dark for 60 min at 37°C.

At the time of sacrifice, mice were sedated and heart punctured with 25 gauge needle and 500–1,000 μl blood removed for WBC analysis using a Coulter Counter (Beckman Coulter, Brea, CA).

To assess toxicity of the different treatments, plasma samples obtained from mice (N=4) in each treatment group at time of sacrifice (11–13 days post-treatment) were tested for standard parameters of liver and renal function, including alanine transaminase (ALT), aspartate aminotransferase (AST), creatinine (Cr), blood urea nitrogen (BUN), albumin and total protein levels.

To assess expression of IFN-γ, IL-2, TNF-α, TGF-β, perforin, granzyme B and FoxP3 genes, ribonucleic acid (RNA) was isolated from tumors using the RNAqueous4PCR kit (Applied Biosystems/Ambion, Austin, TX). The integrity of RNA was tested using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Complementary deoxyribonucleic acid (cDNA) was generated from 5 μg of RNA using Superscript III reverse transcriptase (Invitrogen, San Diego, CA) with oligo-dT as primers according to the manufacturer's protocol. The cDNA (5 μl) diluted 1:40 was used as the template for real-time PCR analysis. The primers and probes (FAM-MGB) for TaqMan-based gene expression assays were purchased from Applied Biosystems (Foster City, CA). Real-time PCR was performed in 384 well thin-wall PCR plates using ABI PRISM 7900HT under the following conditions: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec and a combined annealing/extension step at 60°C for 1 min. Data analysis was performed using SDS 2.21 (Applied Biosystems). The messenger RNA (mRNA) expression level of the target gene was normalized to β-actin using the ΔC_T method. Level of expression=2^−ΔCT, where ΔC_T=C_{T target gene}−C_{T actin}. CT is the cycle threshold at which the fluorescence signal crosses an arbitrary value.

After sacrifice, spleens were removed and each spleen rendered to a single cell suspension using standard techniques (28). Red blood cells were lysed with hypotonic media (ACK lysing buffer, Lonza, Inc. Walkersville, MD) for 3–5 min and then mononuclear cells were washed with PBS two times. Approximately 10⁵–10⁶ cells were stained on ice with Fc block (BD Pharmingen, San Diego, CA) for 15 min, followed by addition of fluorochrome-labeled mAbs to specified cell surface markers for immunophenotyping, and annexin V and propidium iodide for apoptosis analysis. Cells were washed and resuspended and analyzed by a FACSCanto flow cytometer and FlowJo software (BD Biosciences, San Jose, CA).

Sections of frozen tumors (6–8 from each group) were cut to 4 micron thickness. Immediately prior to immunolabeling, sections were re-fixed in cold acetone, equilibrated in PBS-saponin and incubated with 5% normal goat (or donkey) serum for 1 h at 37°C. After washing, they were incubated with rabbit anti-caspase-3 (1:75) for 48–60 h at 4°C, followed by addition of goat anti-rabbit IgG biotin (1:100; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37°C. The anti-caspase-3 Ab detects only the large fragment of activated caspase-3 (17–19 kDa) that results from cleavage after Asp 175 and does not recognize other caspases. Sections were routinely washed in excess PBS, pH 7.5, containing 0.3% saponin (Sigma-Aldrich, St. Louis, MO) at the end of each incubation step, and the same buffer was used as a diluent in all immunohistochemical reagents for caspase-3. These sections were incubated with streptavidin Texas red (1:100; Jackson ImmunoResearch Laboratories) for 1 h at 37°C. Each section was then incubated with rat antibodies to either CD4 or CD8. Sections were then washed with PBS and exposed to goat anti-rat secondary antibodies and pig IgG-FITC (1:50; Jackson ImmunoResearch Laboratories). Sections were then washed and prepared for fluorescence microscopy. Slides were examined with a Nikon TE2,000-U fluorescent microscope (Nikon Corp., Tokyo, Japan). Images were digitized by Photoshop 8.0. Ten high powered fields (×100) from 3–4 slides from each of 6–8 tumors per treatment group were viewed for the presence of fluorescently labeled cells: red (cleaved caspase-3), green (CD4 or CD8 in 2 separate slide sets) or dual-expressing yellow. Numbers of positive cells were counted and mean cell number ± standard error of the mean reported. The individual counting the cells was blinded to the treatment group from which sections were taken.

At the time of sacrifice, splenocytes were isolated and assessed for NK cell cytolytic activity against YAC-1 tumor cells by a standard flow cytometric assay. YAC-1 cells labeled with 3,3′-dioctadecyloxacarbocyanine [DiOC₁₈(3)] purchased from Sigma were cultured with splenocytes for 4 h in triplicate at the effector to target cell (E:T) ratios of 50:1, 25:1, 12.5:1 and 6.25:1. After 4 h, cells were stained with propidium iodide (PI) and washed prior to flow cytometric analysis. Dead target cells were detected as DiOC⁺PI⁺ cells. NK cell activity, defined as percent specific cytotoxicity, was calculated at each E:T ratio using the following formula: (% DiOC⁺, PI⁺ cells) _{Splenocytes+YAC-1} - (% DiOC⁺, PI⁺ cells) _{YAC-1 alone} for each replicate sample. Data were analyzed for significant differences using a linear mixed model to analyze data from 17 mice.

Continuous variables, unless otherwise noted, were analyzed using the Student's t-test. Non-continuous variables (tumor weights) were analyzed using the Mann-Whitney U test. Statistical significance was set at p≤0.05.

To determine if PSK enhances tumor suppression induced by docetaxel, groups of mice (n=10) bearing established TRAMP-C2 tumors were treated with saline control; subtherapeutic doses of docetaxel (5 mg/kg i.p. twice weekly); PSK (300 mg/kg orally by gavage once daily) or a combination of the docetaxel plus PSK (following the same dosing regimens as single agent treatments). A subtherapeutic dose of docetaxel was used to allow observation of enhancing effects of the combination treatment, consistent with previous studies that showed direct antitumor effects of adding intraperitoneally administered PSK to docetaxel treatment in mouse models of gastric cancer (26,27). Doses of up to 1,000 mg/kg PSK have been used in other studies with no reported toxicities (29). The combination of docetaxel plus PSK resulted in significantly smaller tumors, relative to saline control, docetaxel or PSK alone (Fig. 1) (p<0.05), though PSK-treated tumors were not significantly different than control.

In order to assess potential mechanisms by which PSK enhances the effect of docetaxel induced tumor regression, tumors from treated mice were stained for Ki67 (proliferation) and annexin V (apoptosis). Combining PSK with docetaxel resulted in a reduction of Ki67 positive cells compared to docetaxel alone (p<0.05), and induced a 3-fold increase in the number of apoptotic, TUNEL positive cells compared to therapy with PSK (p<0.01), and a 1.5-fold increase compared to docetaxel alone (p<0.05) (Fig. 2).

Given the apoptosis-inducing effects that we observed, we investigated weight, hepatic and renal function in mice treated with PSK or docetaxel alone or in combination. No significant differences in liver and kidney function were observed between the treatment groups (Table I). Interestingly, weight was actually maintained with addition of PSK to the docetaxel regimen as compared to either the control or docetaxel groups (Fig. 3), suggesting a protective effect of the combined treatment against tumor-induced weight loss. Thus, no significant toxicity was observed with the PSK and docetaxel combination therapy. We next asked whether the combined therapy could allay observed immunosuppressive effects of docetaxel.

To determine the impact of PSK on docetaxel-induced immunosuppression, WBC counts were determined. Docetaxel alone induced a 25% reduction in WBC, and addition of PSK decreased this suppressive effect by ~50%. The difference in WBC level observed in response to PSK + docetaxel treatment compared to docetaxel alone was statistically significant (p=0.03) (Fig. 4). Whether this effect is protective (inhibits chemotherapy-induced lymphatic suppression) or restorative (increases WBC levels after therapy) remains to be determined.

Since PSK enhanced antitumor responses in a Her2/neu breast cancer mouse model (17), we investigated whether combining PSK with docetaxel could enhance measures of antitumor immunity in the TRAMP-C2 model. First we asked whether numbers of TILs changed in response to the individual and combined treatments. Tumor sections were stained for expression of tumor infiltrating CD4⁺ and CD8⁺ T cells. The PSK and docetaxel treatments alone resulted in higher numbers of TILs than in control mice, and the addition of PSK to docetaxel increased both tumor infiltrating CD4⁺ T cells compared to PSK or docetaxel alone (p<0.05) and CD8⁺ T cell numbers compared to docetaxel alone (p<0.05) (Fig. 5), suggesting an enhancing effect of the combined treatment on tumor infiltrating T cells.

Given the observation of PSK and docetaxel-induced TIL enhancement, we further analyzed whether any changes in IFN-γ, IL-2, TNF-α, TGF-β, perforin, granzyme B and FoxP3 mRNA expression in the tumor micro-environment could be observed in response to the treatments. TRAMP-C2 prostate tumors from the 4 different treatment groups of mice were assessed using RT-PCR for the presence of mRNA of cytokines and other antitumor immune response markers, as well as for the T-regulatory cell marker, FoxP3. Docetaxel treatment alone did not induce IFN-γ. Significant induction of interferon gamma (IFN-γ) mRNA expression in TRAMP-C2 tumors occurred in mice treated with PSK, either alone (p=0.04) or with docetaxel (p=0.01) compared to saline control (Fig. 6A). No significant differences between the treatment groups were observed for the other markers examined. However, mean IL-2 mRNA expression in response to combined PSK and docetaxel treatment was higher than docetaxel treatment alone (Fig. 6B). Mean levels of tumor necrosis factor-alpha (TNF-α) mRNA in response to docetaxel or PSK treatment alone or in combination trended toward an increase compared to control (p=0.09) (Fig. 6C). No significant differences in perforin, granzyme B, TGF-β or FoxP3 mRNA expression were observed between treatment groups (data not shown).

Since PSK enhanced docetaxel-induced apoptosis of tumor cells, we next investigated whether this effect was specific to tumor cells or if PSK treatment also modulates apoptosis induction of splenic immune cell sub-sets. Immunophenotyping was used to analyze distribution of splenic lymphocyte sub-populations alone and with annexin V, a marker of apoptosis. No significant differences in distribution of splenic T cells, NK cells, B cells or dendritic cells (DC) between treatment groups were observed (data not shown). Further, levels of apoptotic cells among the splenic sub-populations were not significantly different in response to treatments compared to control. Thus, the PSK and docetaxel treatments alone or combined do not appear to induce splenic cell apoptosis.

We attempted to take a similar approach to assess the effect of PSK and docetaxel treatments on apoptosis in tumor-infiltrating T cells. We were unable to disassociate the tumors specimens sufficiently to create single cell suspensions needed for flow cytometry. As an alternative strategy, 8 frozen tumor sections from mice in each treatment group were processed for dual immunohistochemistry, staining for the caspase-3 apoptosis marker and CD4 or CD8 T cell markers. Relatively little apoptosis among CD4 or CD8 cells was observed, regardless of type of treatment (data not shown).

Spleens were collected and preserved from additional mice from each treatment group to assess the influence of PSK alone and combined with docetaxel on NK cell activity against YAC-1 tumor target cells. PSK and docetaxel combination therapy was found to have the greatest NK cell-inducing activity (Fig. 7). The splenic NK cell activity in mice treated with the combination of PSK and docetaxel was significantly higher compared to control mice at the 50:1 E:T ratio (p=0.045).