Microspheres targeted with a mesothelin antibody and loaded with doxorubicin reduce tumor volume of human mesotheliomas in xenografts

The HMESO MM line was previously described [25] and obtained from Joseph R. Testa (Fox Chase Cancer Center, Philadelphia PA). HMESO cells were maintained in Dulbecco’s Modified Eagle Medium DMEM/F12 50/50 (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS), 0.1 μg/mL hydrocortisone (Sigma, St. Louis, MO), 2.5 μg/mL insulin, 2.5 μg/mL transferrin, 2.5 ng/mL sodium selenite (Sigma, St. Louis, MO) and penicillin-streptomycin (50 U/mL penicillin G, 50 μg/mL streptomycin sulfate) (Invitrogen, Carlsbad, CA) [26]. Cells were maintained at 37°C in 5% CO₂.

Porous, amorphous silica microparticles (APMS) pre-modified with TEG on their external surfaces [11], were used for subsequent modifications in experiments described here [13]. APMS microparticles were modified using an antibody to mesothelin (MB) and loaded with DOX as previously described [13, 24].

The SCID mouse xenograft model has been previously described [9, 26]. Figure 1 shows the treatment regimen employed here. Groups consisted of mice receiving saline (0.9% NaCl {pH 7.4}), APMS-MB (no DOX), DOX high (0.2 mg/kg), DOX low (0.05 mg/kg), or APMS-MB-DOX (0.05 mg/kg) (n = 6 mice/treatment group). In brief, 1 × 10⁶ HMESO cells (in 50 μL sterile 0.9% NaCl {pH 7.4}) were injected IP into each mouse. All mice were weighed 3×/wk for 4 wks prior to treatments. Both free-floating spheroid and mesenteric tumors lining the diaphragm develop at 4 wks in all mice [9], at which time IP treatments were initiated. Mice were weighed on each treatment day prior to injection to enable the adjustment of dose per individual body weight. All animal procedures were approved by the University of Vermont IACUC committee.

Mesenteric and spheroid tumors from treated mice were weighed and measured using digital calipers following euthanasia and resection. Free-floating spheroid tumors were weighed individually if possible, but in cases where spheroids were too small, all the spheroids (< 1 mm³) for an individual animal were pooled and measured as one mass. Tumor volumes were calculated following the formula (l × w × h × pi/6). The average tumor volume and weight per mouse was then calculated for each treatment group. We also evaluated the percentage distribution of tumors at different sites (Table 1). Gross examination of tumors and major organs was performed following euthanasia.

Mesenteric and spheroid tumors collected from saline controls and animals treated with DOX low (0.05 mg/kg) or APMS-MB-DOX (0.05 mg/kg) were preserved in 4% paraformaldehyde, paraffin embedded, and processed for hematoxylin and eosin (H&E) staining. Processing and sectioning (4 μm) of tissues was performed in the Department of Pathology (Fletcher Allen Health Care, Burlington, VT). Staining of tissues using H&E was performed following standard protocols [27], and MM tissue sections were examined for necrosis by a board-certified pathologist (KJB). Images were captured using an Olympus BX50 upright light microscope (Olympus America, Lake Success, NY) with an attached Q Imaging Retiga 2000R digital CCD camera (Advanced Imaging Concepts, Inc., Princeton, NJ).

Mesenteric and spheroid tumor sections were de-paraffinized in xylenes (2 × 15 min) and rehydrated to ddH₂O through a graded series of ethanol (100% to 50%). Staining of tissues for Ki-67 as a marker of proliferation was performed following standard protocols as previously described [28]. The monoclonal mouse anti-Human Ki-67 primary antibody (Vector Laboratories, Burlingame, CA, and Leica Microsystems, Buffalo Grove, IL) (1:25 in 1% bovine serum albumin (BSA) in 1× phosphate buffered saline (PBS)) was used [29]. Slides were then dehydrated by dipping them in 100% ethanol (5×) twice, xylenes (5×), and then allowed to air dry before they were coverslipped using Permaslip® (American MasterTech, Lodi, CA) as the mounting agent. Cell proliferation, measured as the percentage of Ki-67 positive cells was quantitated by counting the number of Ki-67 positive (brown nuclei) and negative (purple nuclei) tumor cells, in 5 random Ki-67 expressing regions consisting of 200 cells each. Representative images were captured using an Olympus BX50 upright light microscope (Olympus America, Lake Success, NY) with an attached Q Imaging Retiga 2000R digital CCD camera (Advanced Imaging Concepts, Inc., Princeton, NJ).

To determine patterns of populations of TAMs associated with tumor masses, slides cut from frozen OCT blocks of saline and APMS-MB groups were fixed in acetone for 15 min and stained as described previously (10), but using the primary antibodies, rabbit anti-mouse NOS II (1:1000 dilution) for M1 TAMs and rat anti-mouse CD206 (1:200 dilution) for M2 TAMs. Both antibodies were from AbD, Serotec (Raleigh, NC). The secondary antibodies used were goat anti-rabbit Alexa 555 and goat anti-rat Alexa 488. Both secondary antibodies were diluted 1:500 and purchased from Invitrogen (Carlsbad, CA). Nuclei were stained with 4,′6-diamidino-2-phenylindole (DAPI) (1:200 dilution). All antibody and DAPI dilutions were in PBS/1% BSA. Slides were coverslipped as described previously (10) and examined with a confocal laser scanning microscope (CLSM) Zeiss 510 META (Thornwood, NY). Tiled images (5 images × 5 images) were used to assess profiles of TAMs in whole MMs, and representative areas were examined at higher magnifications (10).

Mice were euthanized with 0.1 mL of Sleep Away (26% sodium pentobarbital, Webster Veterinary) before 5 mL of cold phosphate buffered saline (PBS) (Ca/Mg-free) was instilled into the peritoneal cavity of each mouse using an 18 gauge needle. The abdomen was then lightly massaged, and the PBS removed. PLF was centrifuged, and cell free supernatant was stored at -80°C. Total white blood cell counts in PLF were assessed manually using a hemocytometer. Cytospins were prepared from 50,000 cells following standard protocols [14, 26] and were stained using a HEMA 3 kit (Fisher Scientific, Middletown, VA) per the manufacturer instructions. Three hundred cells per slide were counted by two individuals. The counts were then averaged, and the percentages of each cell type and total numbers were used to calculate the total numbers of cell type/mL for each animal in each treatment group.

After collection of PLF fluid as previously described [9], PLF samples (500 μL) from the saline, DOX low (0.05 mg/kg) and APMS-MB-DOX (0.05 mg/kg) treatment groups were centrifuged at 10,000 × g at 4°C for 5 min. The supernatant was transferred to a fresh Eppendorf vial, and a daunorubicin internal control (final concentration 1 μM) was added to samples. Samples were then vortexed briefly and incubated at 37°C for 15 min in the dark. Proteins were precipitated from PLF samples by adding 250 μL of acetone and 50 μL of aqueous ZnSO₄ solution (400 mg/mL) followed by vortexing. Samples were then centrifuged at 20,000 × g (10 min) at 4°C, and the supernatants were transferred to new vials, and dried under a fume hood in a heat block (65°C) under a stream of nitrogen. The residue was solubilized in 1 mL of methanol, and 200 μL of sample was used for HPLC analysis.

A section of tumor tissue from one tumor/animal from the saline, DOX low (0.05 mg/kg) and APMS-MB-DOX (0.05 mg/kg) treatment groups was removed, and the wet weight recorded. The tissue was then placed in a vial of 380 μL PBS, and 10 μL of lysis solution (Triton X-100, 3% final concentration) was added. Tissue was homogenized using a Biospec Tissue-Tearor (Biospec Products, Racine, WI). Ten μL of Proteinase K (10 mg/mL) was added to the homogenized tissue followed by incubation for 45 min in a water bath at 65°C. The internal standard, daunorubicin, was added to each sample (112.8 μL of 10 μg/mL solution), and samples were brought to a final volume of 800 μL with 1 × PBS. Ten μL of phenylmethylsulfonyl fluoride was added to samples for 10 min. Twenty μL MgCl₂ (0.4 M) and 40 μL DNase I (1 mg/mL) were then added, and samples were centrifuged (10,000 × g) for 5 min at 4°C followed by incubation in a water bath at 37°C for 30 min. Following incubation, 450 μL of each sample was added to 450 μL methanol and 45 μL ZnSO₄ (400 mg/mL), vortexed, and centrifuged (10,000 × g) for 5 min at 4°C, whereupon 200 μL was used for HPLC analysis.

The HPLC method for detection of DOX described previously [9, 30] was followed for the analysis of all samples.

The General Linear Models procedure (PROC GLM) of the SAS System for Windows was used for statistical analysis of data. All data (excluding the analysis of weights over time) were evaluated primarily by one way analysis of variance (ANOVA) followed by Fisher’s LSD pairwise tests for adjustment of multiple pair-wise comparisons between treatment groups. For the weights across time data, a repeated measures analysis of variance was performed on mouse weights to compare the rate of weight loss among treatment groups. Statistical significance was determined as p ≤ 0.05.

We hypothesized that targeted treatment using APMS-MB-DOX would reduce MM tumor volume and weight more effectively than the same dose of DOX administered alone in an established IP xenograft model [9, 26] (Figure 1). After 3 wks of treatment, we observed that mice receiving IP injections of DOX at 0.2 mg/kg (high concentration) had significantly reduced tumor volumes (p ≤ 0.05) compared to saline treated mice, those receiving DOX at 0.05 mg/kg (low concentration), or those receiving APMS-MB without DOX (Figure 2A). Mice receiving APMS-MB-DOX at 0.05 mg/kg also showed reduced tumor volume compared to the same groups (p ≤ 0.05). Consistent with these results, average tumor weight was significantly reduced (p ≤ 0.05) in the DOX high (0.2 mg/kg) and the APMS-MB-DOX low (0.05 mg/kg) groups when compared to saline controls. Treatment with DOX (0.05 mg/kg) alone, or APMS-MB groups did not have an effect on tumor weight in comparison to saline controls (Figure 2B). These results demonstrate that treatment with APMS-MB-DOX (0.05 mg/kg) significantly reduces tumor weight in contrast to the DOX (0.05 mg/kg) group alone. Moreover, they suggest that changes in tumor weight and volume are due to more efficient delivery of DOX and not due to the particles (APMS-MB) themselves. In addition, we evaluated the distribution of MMs and their metastases to distal organs (Table 1). These studies revealed that the vast majority of tumor metastases occurred in the intestines or ovaries/testes (approximately 25% of MMs at each site) in untreated (saline control) mice. These numbers were reduced most dramatically (15 and 11%, respectively) in the APMS-MB-DOX (0.05 mg/kg) group.

Once tumors become large (> 2.0 cm³), MM-bearing mice rapidly lose weight, become lethargic, and cease normal grooming behavior. We hypothesized that modification of the APMS microparticles with a targeting MB would increase drug delivery to tumor tissues and decrease DOX-related weight loss. The weights of mice used in this study were measured throughout the experiment. Indeed, mice treated at the lower, but effective dose of APMS-MB-DOX (0.05 mg/kg) had less average weight loss than those treated with the high dose (0.2 mg/kg) of DOX alone (Figure 3). Pair-wise comparisons of weight loss rates also showed that the treatment group receiving the higher dose of DOX (0.2 mg/kg) had a significantly different slope (*) than other treatment groups. Thus, mice receiving a high dose of DOX alone lost significantly more weight over time in contrast to mice receiving a lower non-effective dose of DOX alone (0.05 mg/kg), or mice receiving APMS-MB-DOX (0.05 mg/kg). These results suggest that an effective dose of APMS-MB-DOX is well tolerated over the duration of multiple treatments.

Gross examination of the tumors and major organs in the peritoneal cavity following euthanasia revealed that mice in all groups had MM involvement with the intestine, reproductive organs, stomach, gut, kidney, liver and spleen (Table 1). Animals treated with DOX high (0.02 mg/kg) or DOX low (0.05 mg/kg) also had bloody PLF. Bloody PLF was not observed in mice treated with APMS-MB-DOX (0.05 mg/kg). Given the reduction in tumor volume, decreased weight loss, and the lack of bloody PLF in groups treated with APMS-MB-DOX (0.05 mg/kg) in comparison to the same dose of DOX alone, APMS-MB or saline controls, we hypothesized that tumor tissue from these animals would have increased areas of necrosis and fewer proliferating cells. We stained representative mesenteric and spheroid (free-floating) tumors [9] from each animal using H&E staining in the saline, DOX low (0.05 mg/kg) and APMS-MB-DOX (0.05 mg/kg) treatment groups, and a board-certified pathologist assessed the tissues to evaluate the average percentage of total tumor area exhibiting necrosis. Examination of mesenteric tumors revealed more necrotic tissue in the saline treatment group - an average of 65% compared to the DOX (0.05 mg/kg) alone (53%) or APMS-MB-DOX (41%) groups. However, these trends were not statistically significant. In spheroid tumors, necrosis was roughly comparable between treatment groups (20-25%) and lower overall (Figure 4A, B). No significant changes in the percentage of Ki-67-positive, i.e., proliferating cells, were observed in mesenteric MMs (Figure 5A-C). However, Ki-67-positive cell staining in spheroid MMs showed significantly decreased proliferation in the APMS-MB-DOX (0.05 mg/kg) treatment group when compared to saline controls or mice receiving a comparable dose of DOX alone (0.05 mg/kg) (p ≤ 0.05). Histopathology of spheroids showed focal areas of necrosis in the interiors of these smaller, ascites-like tumors. Thus APMS-MB-DOX may have a greater impact on inhibition of proliferation because microparticles are in the IP milieu and penetrate and access the tumor interior more easily [10].

We have speculated, based on many findings, that chronic inflammation is a feature of the development and establishment of MMs [31]. To address this question, and to determine whether or not the treatment regimens used in these experiments affected immune cell profiles in PLF, cytospins were evaluated as described previously [9]. PLF samples from treatment groups receiving effective concentrations of APMS-MB-DOX (0.05 mg/kg) or DOX alone (0.2 mg/kg) contained a significantly higher (p ≤ 0.05) total number of cells/mL in PLF than mice treated with DOX (0.05 mg/kg) alone or APMS-MB (Figure 6A). Differential cell counts revealed that when compared to the saline group, the percentage of macrophages was significantly higher in all treatment groups, whereas numbers of neutrophils were significantly lower (p ≤ 0.05) (Figure 6B).

To confirm that the reduction in tumor volume and weight was due to DOX, we examined levels of DOX in tumor homogenates in tissues and PLF fluids of tumor-bearing mice receiving 3× weekly doses of DOX (0.05 mg/kg) or APMS-MB-DOX (0.05 mg/kg). HPLC analysis was performed at the end of 3 wks of treatment (data not shown). We were unable to detect DOX in the PLF of either treatment group, suggesting it had been released and already excreted and/or accumulated in tumor tissue. Supporting the latter hypothesis, DOX was detected in the tumor tissues of treatment groups receiving DOX alone (2.47 nmoles/gm of MM tissue) or APMS-MB-DOX (2.57 nmoles/gm of MM tissue). Though the amount of DOX was slightly higher in the MMs in the targeted therapy group, the amounts of DOX were not significantly different between groups.

MMs are historically associated with areas of inflammation in both animal models and human tissues, and TAMs also are a prominent feature of our SCID mouse model [10]. In studies here, our objective was to determine if TAMs in MMs reflected M1 (anti-tumor) and/or M2 (pro-tumor) phenotypes. In both untreated (saline) and APMS-MB mice, M2 (green) TAMs appeared to predominate (Figure 7), but M1 TAMs (red) were noted in discrete surface accumulations along the edges of tumors. M2 were found more frequently in the interior of tumors. Further quantitative studies are planned in all treatment groups.