Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms

Abstract

Podoplanin (PDPN) is a unique transmembrane receptor that promotes tumor cell motility. Indeed, PDPN may serve as a chemotherapeutic target for primary and metastatic cancer cells, particularly oral squamous cell carcinoma (OSCC) cells that cause most oral cancers. Here, we studied how a monoclonal antibody (NZ-1) and lectin (MASL) that target PDPN affect human OSCC cell motility and viability. Both reagents inhibited the migration of PDPN expressing OSCC cells at nanomolar concentrations before inhibiting cell viability at micromolar concentrations. In addition, both reagents induced mitochondrial membrane permeability transition to kill OSCC cells that express PDPN by caspase independent nonapoptotic necrosis. Furthermore, MASL displayed a surprisingly robust ability to target PDPN on OSCC cells within minutes of exposure, and significantly inhibited human OSCC dissemination in zebrafish embryos. Moreover, we report that human OSCC cells formed tumors that expressed PDPN in mice, and induced PDPN expression in infiltrating host murine cancer associated fibroblasts. Taken together, these data suggest that antibodies and lectins may be utilized to combat OSCC and other cancers that express PDPN.

Keywords: cancer; cell migration; lectin; podoplanin; receptor.

Figure 1. PDPN expression in OSCC cells

PDPN expression in OSCC cells was examined by immunohistochemistry. (a) Specimens from oral cancer patients (bar = 40 microns). (b) Cultured OSCC cells (bar = 100 microns).

Figure 2. Pdpn expression correlates with OSCC cell motility

(a) PDPN and GAPDH were detected by Western blotting of protein (20 μg per lane) from HSC-2, HSC-4, and HSQ-89 OSCC cells. (b) PDPN expression was quantitated by image densitometry and shown as mean+SEM (n=2). (c) Cell migration was evaluated by wound healing and quantitated as the number of cells that migrated into a 200 × 300 micron area in the center of the wound at 18 hours (mean+SEM, n=5). Single, double, and quadruple asterisks indicate p<0.05, p<0.01, and p<0.0001 compared to HSC-2 cells, respectively.

Figure 3. Reagents that target PDPN can decrease OSCC cell migration and viability

(a) Wound healing migration assays were performed on confluent OSCC monolayers treated with concentrations of NZ-1 or MASL as indicated. Data are shown as the number of cells that migrated into a 200 × 300 micron area along the center of the wound in 18 hours (mean+SEM, n=5). (b) NZ-1 and MASL toxicity was evaluated by Trypan blue staining of cells treated with NZ-1 or MASL for 24 hours and quantitated as the number of living cells in a 3 mm² field (mean+SEM, n=5).

Figure 4. Effects of reagents that target PDPN on GTPase activity

Active RhoA, Rac1, and Cdc42 GTPase was detected in HSC-2 cells treated with 0 nM, 770 nM, and 2310 nM NZ-1 or MASL as indicated. Data are shown as percent control untreated cells (mean+SEM, n=3).

Figure 5. Effects of NZ-1 and MASLon OSCC cell morphology

OSCC cells were treated with 2310 nM NZ-1 or MASL for 24 hours and examined by microscopy as indicated (bar = 100 microns).

Figure 6. NZ-1 and MASL do not induce caspase or PARP cleavage in OSCC cells

(a) PARP, Caspase 8, and GAPDH were examined by Western blotting of protein from OSCC cells treated for 24 hours with 0 nM (control) or 2310 nM NZ-1 or MASL as indicated. (b) Signal was quantitated by image densitometry and shown as the percent of cleaved PARP and Caspase 8 compared to total PARP and Caspase 8, respectively (mean+SEM, n=3).

Figure 7. Mitochondrial membrane permeability transition inhibition protects cells from NZ-1 and MASL toxicity, while caspase inhibition does not

The effects of the pan-caspase blocker Z-VAD-FMK and the mitochondrial membrane permeability transition blocker cyclosporin A were examined on HSC-2 and HSC-4 cells treated with 2310 nM NZ-1 or MASL as indicated. Data are shown as percent of cells killed (mean+SEM, n=6).

Figure 8. NZ-1 and MASL exhibit different OSCC cell binding dynamics

(a) HSC-2 cells were incubated with fluorescently labeled MASL or NZ-1 for 2 minutes and examined by confocal microscopy. MASL bound to cell membranes, while NZ-1 did not. (b) Colocalization of MASL and PDPN is evident by confocal microscopy. Bars = 50 microns.

Figure 9. MASL inhibits melanoma and OSCC cell metastasis in zebrafish embryos

(top) DiI labeled B16 melanoma and HSC-2 OSCC cells were implanted into the perivitelline cavity of zebrafish embryos grown with or without 770 nM MASL as indicated. Tumor cells (red) and blood vessels (green) were visualized after 3 days of growth. (bottom) Metastasis was quantified as the number of tumor cells that moved anterior to the anal opening (indicated by white arrows). Data are shown as mean+SEM. Single and triple asterisks indicate p<0.05 and p<0.001 (n≥20).

Figure 10. PDPN expression in human OSCC cells and infiltrating mouse fibroblasts in xenograft tumors

Tumors from HSC-2 cells were examined with antisera specific for human (D2-40) and mouse (8.1.1) PDPN by immunohistochemistry as indicated.