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Reviewer #1 Evaluations:
Recommendation: Accepted, but needs revision
Priority for Publication (1 is high) : 2
Helsinki violation questions?: No
Reviewer #1 (Comments to the Author):
The authors demonstrate an innovative strategy to deliver substances to tumour cells by
manipulation/purification of mesenchymal stem cells. The powerful lentiviral-based technique is
functional tested in nude mice orthotopic grafted with pancreatic tumour. The manuscript is well
written, the figures are adequate.
Major Comments
1. The authors demonstrate the method as powerful tool for basic research and improvement in
gene therapy. This second point should be critical discussed and further addressed in the section
Discussion. Valid data supporting this point of view are not given in the present version of the
manuscript.
Our response: the thank the reviewer for this valuable hint.
Change: We further discussed this point and added new references – see first marked paragraph
of the "Discussion" section.
2. The authors nicely demonstrate that neither LV-transduction nor puromycin selection
compromise MSC phenotype or multipotency. It has been shown that CD34- stem cells can give
rise to CD34+ cells, which obtain this phenotype while circulating in the peripheral blood (PNAS
1995,92;748-752). Is there any data available in the model presented? Is there any possibility to
perform additional (co-) immunostainings?
Our response: the thank the reviewer for the valuable hint. The reviewer refers to the known
heterogeneity of MSC cultures, due to the lack of a single definitive marker for MSC. MSC are
just defined by three criteria: 1. Plastic adherence. 2. Positive for expression of CD105, CD73,
CD90. 3. Lack of expression of haematopoietic antigens like CD45, CD34 and markers for
monocytes, macrophages and B cells. In the PNAS 1995 publication the reviewer mentioned, the
authors used canine bone marrow cells with hematopoietic characteristics. These cells cannot
compared to our used human bone marrow derived MSC and we did not found comparable
results in our experiments. Also, we ensured by several control experiments, that isolated MSC
behave their CD34- phenotype even after several rounds in culture - compare our recent enclosed
publication by Wagner et al, 2006. In addition, we cultured the MSC in medium for
hematological cells (RPMI, 10% FCS or Stemline II Medium, Thrombospondin [TPO 80 ng/ml],
stem cell factor [SCF, 50 ng/ml], FL-3 ligand [50 ng/ml] and did not found differentiation of the
MSC to a CD34+ phenotype – compare our recent enclosed publication by Wagner et al, 2007.
Change: We added the above described control experiments of our former studies to the
"Material and Methods" section – see marked text. The two "Wagner et al." references are cited.
- Wagner, W., Feldmann, R.E., Jr., Seckinger, A., Maurer, M.H., Wein, F., Blake, J., Krause, U., Kalenka,
A., Burgers, H.F., Saffrich, R., Wuchter, P., Kuschinsky, W. and Ho, A.D. (2006) The
heterogeneity of human mesenchymal stem cell preparations--evidence from simultaneous
analysis of proteomes and transcriptomes. Exp Hematol, 34: 536-548
- Wagner, W., Wein, F., Roderburg, C., Saffrich, R., Faber, A., Krause, U., Schubert, M., Benes, V.,
Eckstein, V., Maul, H. and Ho, A.D. (2007) Adhesion of hematopoietic progenitor cells to human
mesenchymal stem cells as a model for cell-cell interaction. Exp Hematol, 35: 314-325
3. It is well-known that adult stem cells contribute to tissue regeneration partly by promoting
neovascularization or arteriogenesis. However, induction of vascularization is suggested as one
important mechanism in tumour growth. Did the authors analyse vascular structures
accompanying the MSC-tumours?
Our response: Yes, we agree that not genetically manipulated MSCs may promote angiogenesis
and tumor growth. Further genetic engineering is required to provide MSC with therapeutic
genes and to avoid by this way a contribution of the MSC vehicles to angiogenesis and tumor
growth. We are currently designing LV vectors delivering antiangiogenic genes to specifically
target tumor vasculature. However, these data are too preliminary to be shown in the actual
paper submitted to Cancer Gene Therapy.
Change: This point is discussed – see first marked paragraph of the "Discussion" section.
4. For orthotopic tumour growth, cell clots were mechanically incorporated which is associated
with tissue damage. How do the authors rule out the possibility that migration of MSCs into
tumours is not induced by regenerative tissue remodelling (wound healing/ tissue remodelling
might persists over 3-4 weeks after injury)?
Our response: Yes, we agree that wounds may attract MSC. However, there is evidence against
migration of MSC to the wound we made by xenografting. This is related to the technique of the
mouse model, the time point of MSC injection and the fact that we did not found MSC in normal
pancreatic tissue near the implantation side. First, the orthotopic tumor model used by us is not
associated with severe tissue damage since only a small tissue pocket is generated using a
microscissor and blunt parenchymal dissection. In this pocket a very small tumor fragment from
a subcutaneous tumor is inserted without any need to further anchor the tumor transplant since
the tissue texture will cause spontaneous closure of the pocket. It is also noteworthy that there is
virtually no bleeding in the implantations procedure.
Secondly, MSCs were injected after a
period of 4 weeks of tumor growth. At this time point there should be no active wound healing
due to the implantation process. Tissue remodelling might be due to ongoing rapid tumor
expansion in the pancreas rather than related to the primary tumor inocculation. This is also of
importance since in this study MSC require a functional vascular network which is triggered by
the tumor. Nevertheless, the reviewers comment is correct since ongoing tumor growth certainly
leads to local tissue remodelling and adaptive processes including neoangiogenesis,
lymhovascular development and establishment of the specific tumor stroma. Finally, we show
that MSC did not home to normal pancreatic tissue close to a putative wound, but specifically
into the tumor xenograft. This speaks against the theory of homing to wound/tissue remodeling
based on the initial implantation surgery.
Change: This point is discussed – see second paragraph with marked text in the "Discussion"
section.
Minor Comments
5. Equal contribution of first AND senior authors is declared. This should be justified in a short
statement to the EDITOR of CGT.
Contribution of first authors:
1. G. Kallifatidis: made experiments shown in Fig. 1, 4, 5
2. B. Beckermann: made experiments shown in Fig. 1, 6
3. Ariane Groth: made experiments shown in Fig. 1, 2, 3
4. Mario Schubert: wrote the animal application which is the basis for Fig. 6, provided MSC,
suggested experimental procedures.
Contribution of last authors:
5. Peter Büchler: had the initial idea for the study, was involved in conceptional design of
experiments and supervision of first authors.
6. Ingrid Herr: was involved in conception of the study, suggested experimental procedures,
supervised the experiments, wrote the paper and prepared the figures.