Multifaceted immunotherapeutic effects of vitamin D-binding protein-derived

macrophage activating factor (GcMAF) on human breast cancer cells

Pacini S*, Ward E**, Smith RJ**, Thyer L**, Gulisano M*, and Ruggiero M***

*Dept of Exp and Clin Med, Firenze Italy**Macro Innovations Ltd, Innovation Park, Cambridge, UK

***Dept of Exp and Clin Biomed Sci, Firenze, Italy.

Introduction

• Breast cancer is the most common type of cancer in women and one of the most common causes of cancer death.

• This leads to the continuous search for additional therapies that could be used as a complement to or alternative to surgery, radiation therapy and conventional chemotherapy.

• Among these novel approaches, those that target the immune system (often referred to as immunotherapy) and tumour-induced angiogenesis, appear promising.

• Vitamin D-binding protein-derived macrophage-activating factor (DBP-MAF, also known as GcMAF) is a good candidate for both immunotherapy and antiangiogenic therapy since it stimulate macrophages, and inhibits angiogenesis.

• The impressive results of GcMAF on 16 non-anaemic women who received weekly administration of GcMAF as treatment for metastatic breast cancer were recently described.

Materials and Methods

• Here we describe the effects of GcMAF (First Immune GcMAF by Immuno Biotech Ltd ) on human breast cancer cells (MCF7 by HPA Culture Collection).

• Proliferation, morphology, vimentin expression and angiogenesis were studied by cell proliferation assay, phase-contrast microscopy, immunohistochemistry and western blotting.

• The effect of GcMAF on tumor-induced angiogenesis was studied by chorioallantoic membrane (CAM) assay.

• The effects of GcMAF in activating tumoricidal macrophages was studied by time-lapse microphotography (NCH Debut capture software).

Experimental Results

GcMAF inhibits breast cancer cell proliferation

Cells were seeded at semi-confluence in medium containing 1% FCS and 0.4 ng/ml Gc-protein (column 1, control), or GcMAF (column 2). Cells were counted after 72 h. Results are expressed as means+SEM (n=4).

Maximal inhibitory effects on cell proliferation were observed with 0.4 ng/ml GcMAF concentration. This concentration was similar to that required to stimulate human peripheral blood mononuclear cells (Cancer Immunol Immunother 60:479-85, 2011).

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Phase contrast light microscopy of MCF-7 living cells. Cells did not undergo any treatment, i.e. washing, fixation or staining. Magnification 300x.

Upper panel; cells treated with 40 ng/ml Gc-protein for 72 h. Cells did not show contact inhibition and formed tumour clusters.

Lower panel; cells treated with 40 ng/ml GcMAF for 72 h. Cells grew in monolayer and no clusters could be observed. Cells were regularly polygonal and uniform in morphology and size.

GcMAF causes reversion of the neoplastic phenotype.

Typical cancer cell morphology and behaviour

Typical normal cell morphology and behaviour

• GcMAF-induced morphological changes can be interpreted as if DBP-MAF reverted cancer cell malignant phenotype, a phenomenon confirmed by the study of vimentin expression.

• Vimentin expression is considered a hallmark of human breast cancer progression. In fact, during progression toward a more malignant phenotype, the cell intermediate filament status changes from a keratin-rich to a vimentin-rich network in a process termed “epithelial-mesenchymal transition” (Cells Tissues Organs 185:191-203, 2007).

Strong vimentin expression (brown) in MCF-7 cells treated with 40 ng/ml Gc-protein (control) for 72 h. Immunohistochemical analysis, 600x.

GcMAF and vimentin expression

• Immunohistochemical analysis, demonstrated that exposure to 40 ng/ml GcMAF for 72 h significantly decreased vimentin expression in MCF-7 cells.

• Brown staining was less pronounced and cells also appeared more uniform in morphology and size, and more adherent to the substrate. Densitometric analysis confirmed these observations.

• These data, confirmed by western blot analysis, are consistent with GcMAF-induced reversal of epithelial-mesenchymal transition.

• Cancer cells revert to normal.

GcMAF

Control

Control GcMAF

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GcMAF

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GcMAF

GcMAFControl

Western Blot

Densitometric assay

GcMAF-induced morphological changes indicating that GcMAF reverted cancer cell malignant phenotype are corroborated by analysis of vimentin expression.

The asterisks indicate the new blood vessels surrounding the sponge where the human breast cancer cells had been implanted. E: no addition. F: GcMAF, 1 ng/ml. The reduction of the number of blood vessels is impressive.

GcMAF inhibits tumor cell-induced neoangiogenesis

Inhibition of tumour angiogenesis

deprives cancer cells of their energy/blood supply and re-directs energy/blood supply to normal tissues fighting cancer cells.

Starved and weakened cancer cells thus become easy prey of tumoricidal macrophages activated by GcMAF.

The following pictures from a video show activated macrophages “attacking” and destroying a culture of human breast cancer cells.

In the sequence of pictures it can be observed that the irregular growth of the breast carcinoma cells is arrested and the large cell biomass is reduced and carcinoma cells destroyed by ingestion by the GcMAF-activated macrophages.

Macrophages

Human breast cancer cells

When viewed under the microscope, macrophages appear as round spherical cells that refract light and surround cancer cells.

Human breast cancer cells appear adherent to the plate and show the irregular morphology that is typical of cancer cells.

Human breast cancer cells surrounded by GcMAF-activated macrophages collapse in clusters that are further destroyed by macrophages. The pseudo-organized layer of cancer cells that was adherent to the plate is almost disappeared and large parts of the field are now devoid of cancer cells.

Further dramatic reduction of cancer cells. Most of the field is now devoid of cancer cells. Remaining cancer cells are grouped in a cluster that is progressively destroyed by the GcMAF-activated macrophages.

Conclusions

• The biological effects of GcMAF were first described in 1994 and shortly afterward its anti/tumour properties in BALB/c mice bearing Ehrlich ascites tumour were described.

• Further studies on the effects of GcMAF on cancer patients reported successful immunotherapy of prostate cancer, and metastatic breast and colon cancer.

• Here we demonstrate that the anti/cancer efficacy of GcMAF can be ascribed at least to three biological properties of the molecule:

• i) activation of tumoricidal macrophages

• ii) inhibition of tumour-induced angiogenesis

• iii) direct inhibition of cancer cell proliferation, migration and metastatic potential.

• Our results support and reinforce the hypothesis that GcMAF is a molecule endowed with multiple biological activities underlying its powerful anti/cancer properties.

• It is auspicable that GcMAF might soon enter therapeutic protocols for the immunotherapy of human cancer.