Download (GcMAF) on human breast cancer cells

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 macrophageactivating 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 nonanaemic 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
35
Cell number (x10 4)
33
31
29
27
25
23
1
2
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).
Typical cancer cell morphology and behaviour
GcMAF causes reversion of
the neoplastic phenotype.
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.
Typical normal cell morphology and behaviour
GcMAF and vimentin expression
• 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.
Strong vimentin expression (brown) in MCF-7 cells
treated with 40 ng/ml Gc-protein (control) for 72 h.
Immunohistochemical analysis, 600x.
• 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).
Control
GcMAF
• 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 GcMAFinduced reversal of epithelialmesenchymal transition.
• Cancer cells revert to
normal.
Control
GcMAF-induced morphological
changes indicating that GcMAF
reverted cancer cell malignant
phenotype are corroborated by
analysis of vimentin expression.
GcMAF
Western Blot
Control
Control
GcMAF
Control
Densitometric assay
GcMAF
GcMAF
GcMAF inhibits tumor cell-induced neoangiogenesis
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.
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.
When viewed under the microscope, macrophages appear as round
spherical cells that refract light and surround cancer cells.
Macrophages
Human breast 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.