CSPG4 as a target of antibody-based immunotherapy for malignant mesothelioma

Abstract

Purpose: Malignant mesothelioma (MM) is an aggressive cancer, resistant to current therapies. Membrane chondroitin sulphate proteoglycan 4 (CSPG4), which has been successfully targeted in melanoma and breast cancer, was found highly expressed in MM, but not in normal mesothelium. Therefore, we explored CSPG4 as a suitable target for monoclonal antibody (mAb)-based immunotherapy for MM.

Experimental design: We assayed adhesion, motility, invasiveness, wound-healing, apoptosis, and anchorage-independent growth of MM cells on cell cultures. CSPG4 expression and signaling was studied by immunoblotting. The growth of MM severe combined immunodeficient (SCID) mice xenografts induced by PPM-Mill cells, engineered to express the luciferase reporter gene, was monitored by imaging, upon treatment with CSPG4 mAb TP41.2. Animal toxicity and survival were assayed in both tumor inhibition and therapeutic experiments.

Results: CSPG4 was expressed on 6 out of 8 MM cell lines and in 25 out of 41 MM biopsies, with minimal expression in surrounding healthy cells. MM cell adhesion was mediated by CSPG4-dependent engagement of ECM. Cell adhesion was inhibited by mAb TP41.2 resulting in decreased phosphorylation of focal adhesion kinase (FAK) and AKT, reduced expression of cyclin D1 and apoptosis. Moreover, mAb TP41.2 significantly reduced MM cell motility, migration, and invasiveness, and inhibited MM growth in soft agar. In vivo, treatment with mAb TP41.2 prevented or inhibited the growth of MM xenografts in SCID mice, with a significant increase in animal survival.

Conclusion: These results establish the safety of CSPG4 mAb-based immunotherapy and suggest that CSPG4 mAb-based immunotherapy may represent a novel approach for the treatment of MM.

Figure 1. CSPG4 is expressed in Malignant Mesothelioma

A. Mesothelioma cells PPM-Mill, Hmeso and REN (5×10⁴) were harvested with 1mM EDTA and then incubated with CSPG4-specific mAbs 225.28, 763.74, TP32, TP41.2 and TP61.5 for 1 hour at 4°C and with RPE-conjugated goat anti-mouse secondary antibodies for 30 minutes at 4°C. Cells were then analyzed with the FACScan flow cytometer. Results are expressed as percentage of stained cells and as mean fluorescence intensity. The CSPG4-positive melanoma Colo38 cells and the CSPG4-negative Burkitt’s lymphoma cells Raji were used as positive and negative controls, respectively. B. Sections of formalin fixed, paraffin embedded tissues. A CSPG4-positive melanoma lesion was used as a positive control, normal pleura, sarcomatoid MM, biphasic MM and epithelioid MM were sequentially incubated with CSPG4-specific mAb D2.8.5 overnight at 4°C and with peroxidase-conjugated goat anti-mouse IgG antibodies for 1 hour at room temperature. “Epithelioid (CSPG4+)” indicates the epithelioid tumors that stained positive for CSPG4 and “Epithelioid (CSPG4−)” indicates the same type of tumors that stained negative for CSPG4. (Magnification 400X).

Figure 2. CSPG4-specific mAbs inhibit MM cell growth by blocking cell adhesion

A. CSPG4-positive MM cells were plated in serum-free condition on dishes pre-coated with CI, CIV and FN and cultured for 30 minutes. Then cells were stained with crystal violet. Acetic acid was then added to solubilize the cells and the absorbance was measured at 560nm to determine the extent of cell adhesion. Values represent the mean ± SEM of triplicates from three independent experiments. Asterisks indicate P<0.05. B. PPM-Mill MM cells were grown on fibronectin-coated dishes for up to 2 hours. Cell lysates were tested in Western blotting with phosphoFAK-, FAK-, phosphoAKT-, AKT- and Cyclin D1- specific antibodies. GAPDH was used as a loading control.

Figure 3. CSPG4-specific mAb TP41.2 reduces migration and invasion of MM cells

A. PPM-Mill and Hmeso MM cells were pre-incubated with mAb TP41.2 or with IgG control for 1 hour. Cells were then plated on the membrane of the upper chamber of a Transwell^® plate and grown for 48 hours in the presence of 10% FBS. Cells grown in serum-free conditions were used as a negative control. The cells migrated to the lower surface of the membrane were stained with Giemsa staining for microscopical observation. Then cells were solubilized and the absorbance was measured at 560nm to determine the extent of cell migration. The graph indicates the mean ± S.D. from three separate experiments. The statistical differences represent comparisons versus untreated cells using Students’s t test. P ≤ 0.05. B. PPM-Mill, Phi and REN MM cells were pre-incubated with mAb TP41.2 or with IgG control for 1 hour. Cells were then plated on the membrane of the upper chamber of a Matrigel^®-coated Transwell^® plate and grown for 48 hours in the presence of 10% FBS. Cells grown in serum-free conditions were used as a negative control. The cells migrated to the lower surface of the membrane were stained with Giemsa for microscopical observation. The graph indicates the mean ± S.D. from three separate experiments. The statistical differences represent comparisons versus untreated cells using Student’s t test. P ≤ 0.05.

Figure 4. CSPG4-specific mAb TP41.2 reduces the size and growth rate of MM colonies in soft agar

A. CSPG4-positive PPM-Mill and, B. CSPG4-negative REN MM cells were pre-incubated with IgG control or mAb TP41.2 then plated in soft agar. Thereafter, mAb TP41.2 or IgG control mAb were added to the cultured cells every 48 hours. The rate of cells growth was evaluated by phase-contrast microscopy 17 days later. The average number of colonies was quantified by colony counter using Quantity One software analysis and is reported in the bar graph. The size of the colonies was determined in 5 random optical fields from each plate.

Figure 5. CSPG4-specific mAb TP41.2 induces apoptosis of MM cells

CSPG4-positive PPM-Mill and CSPG4-negative REN MM cells were pre-incubated with mAb TP41.2 or IgG control for 1 hour and then cultured for 24 hours on uncoated dishes. Then the extent of apoptosis was evaluated by flow cytometry using Annexin V and 7-AAD. The percentages of live cells, of early and late apoptotic cells and of necrotic cells are indicated in the cytograms. The total percentage of apoptosis is reported in the bar graph. Asterisks indicate p<0.0001.

Figure 6. CSPG4-specific mAb TP41.2 inhibits human MM cell growth in xenografts

CSPG4-positive PPM-Mill/luc cells (1×10⁶/mouse) were pre-incubated with mAb TP41.2 or IgG control for 1 hour and injected i.p. in SCID mice, treatment started at D0 (A) or injected i.p. in SCID mice to induce tumor establishment, treatment started at D5 (D). mAb TP41.2 was administered i.p. twice a week for four weeks. The size of tumors was monitored weekly by IVIS^™ after injection of luciferin. The luminescence signals are expressed as total flux of photons/sec (A: inhibition of MM growth, and D: therapy of MM). Quantitative analysis of the whole-body total photon counts of IgG control and mAb TP41.2 treated mice at doses of 0.2 mg in 200ul per 20g mouse (B: inhibition of MM growth, and E: therapy of MM). Kaplan-Meier survival plots of mice with MM xenografts. Survival curves of xenograft-bearing mice treated with CSPG4-specific mAb TP41.2 or isotype IgG control. In both survival experiments mice were euthanized and necropsied when tumor development caused severe ascites limiting the animal’s mobility, according to IACUC regulations. No animals were censored in both Kaplan-Meier plots (C: inhibition of MM growth, and F: therapy of MM).