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Title:Monitoring of effects of multi-modal triple therapy on tumor invasion andangiogenesis using Contrast enhanced ultrasound (CEUS).
Authors:
Philipp M Paprottka ([email protected])Svenja Roßpunt ([email protected])Michael Ingrisch ([email protected])Clemens C Cyran ([email protected])Konstantin Nikolaou ([email protected])Maximilian F Reiser ([email protected])Brigitte Mack ([email protected])Olivier Gires ([email protected])Dirk A Clevert ([email protected])Pamela Zengel ([email protected])
Version:3Date:1 March 2015
Author's response to reviews: see over
Dear Professors,
We would kindly ask you to consider the enclosed original manuscript entitled
“Reducing tumor growth and angiogenesis using a triple therapy measured with
Contrast-Enhanced Ultrasound (CEUS) for publication in BMC Cancer.
Our previous ex-vivo dates have already been published in your journal (BMC
Cancer. 2010 Mar 11;10:92. doi: 10.1186/1471-2407-10-92. Multimodal therapy for
synergic inhibition of tumour cell invasion and tumour-induced angiogenesis).
Thank you for considering our manuscript into BMC Cancer.
We have addressed all reviewers' comments and have changed our manuscript
accordingly, please see below.
Kind regards,
Philipp Paprottka
Reviewer 1:
1. Minor essential revisionsspelling error on line 59 of the abstract, "profiferation"
According to the reviewer request we changed the spelling. We are very sorry for our
mistake.
2. Discretionary Revisions Is there an orthotopic hypopharynx carcinoma xenoraft
model?
To our knowledge there is no existing orthotopic hypopharynx carcinoma xenoraft
model for rats. An orthotopic hypopharynx carcinoma xenoraft mouse model exists, but
due to the much more smaler animal/tumor size this model is not appropriate for our
measurements and thus not applicable.
3. Discretionary Revisions
Is there data/supplimental data for the effects of Mesupron, Galardin, and Celecoxib
alone to compare with control and triple therapy?
In our previous manuscript we use Mesupron, Galardin and Celecoxib in a single, double
and triple combination therapy. “Untreated FaDu and HeLa cells were characterised by a
strong invasion capacity into fibroblast spheroids, which was set to 100% for the
purpose of comparison (Figure 2). Next, FaDu and HeLa cells were treated with
Celecoxib®, Galardin®, WX-UK1, and combinations thereof, before incubation with
fibroblast spheroids. Celecoxib®, Galardin® displayed no major effect on tumour cell
invasion when applied as single drugs and at the concentration chosen, while the
combination of both inhibitors resulted in a 30% reduction for FaDu cells (Figure 2A
and 2C). WX-UK1 inhibited tumour cell invasion by 50% and 30% in FaDu and HeLa
cells, respectively (Figure 2A and 2C). However, the most effective inhibition was
achieved with a triple combination of WX-UK1, Galardin®, and Celecoxib® with 80%
and 90% of inhibition for FaDu and HeLa cells, respectively (Figure 2). “
Effects of WXUK-1 (SPI), Celecoxib (COXI), and Galardin (MMPI) on FaDu and HeLa
cell invasion capacity. (A) FaDu cells (3000 cells) or (C) HeLa cells (8000) were
cultured in absence or presence of single, double or triple combination therapy. Shown
is the mean infiltration of tumour cells with standard deviation into 15-18 pre-formed
fibroblast spheroids performed in three independent experiments. (B) FaDu and (C)
HeLa single cells (brown staining) and human primary skin fibroblast spheroids (1 ×
104 cells, blue staining) were visualised by immunohistochemistry with antibodies
against cytokeratines and MAb 5B5 in kryosections at day three of co-culture in the
absence (left sections) and in the presence of inhibitors (right sections).
Zengel et al. BMC Cancer 2010 10:92 doi:10.1186/1471-2407-10-92
Level of interest:An article of importance in its field
Quality of written English:Acceptable
Statistical review:No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests:I declare that I have no competing interests
Reviewer 2:
The authors of this study present a series of data which address two primary
hypothesis: 1) that NP carcinomas can be effectively treated with "triple therapy" (a
COX-2 inhibitor, an MMP inhibitor, and a uPA inhibitor), and 2) that changes in tumor
physiology, specifically angiogenesis, can be followed using a modified ultrasound
technique called flash replacement.
There is certainly a degree of novelty in the combination therapy proposed and
especially in the concept of monitoring physiologic changes within the tumor by
noninvasive methods, and the authors should be credited for this. However, there are
major problems with the study in its current form. These are addressed below.
MAJOR COMPULSORY REVISIONS
The language of the manuscript is often casual and imprecise. The report should be
revised to present data and concepts in a more precise way. In addition, there are a large
number of misspellings, typos, and inconsistencies in the document that make it look as
if it were hurriedly or carelessly written. The manuscript would also benefit from review
by a native english speaker, as there are several sentences which are very difficult to
understand. Phrases such as “reduction of tumor size increase” are ambiguous and
difficult to understand.
According to the reviewer request we have re-edited the entire manuscript, including
re-review by two native speakers.
The authors provide inadequate and cursory rationale for combining these specific
therapies into a multi-agent regimen. This should be discussed logically and/or
compared to individual treatments. The concept is suggested that they work in a
synergistic manner, though this cannot be deduced from the data presented or by
reference from papers cited.
In our previous manuscript we analyzed single, double and triple therapy of the
different drugs and compared the effect of them in vitro in a sphäroid and matrigel
model see “Zengel et al. BMC Cancer 2010 10:92 doi:10.1186/1471-2407-10-92”. We
illustrated that the triple combination is much more effective in comparison to the single
or double combination (see Figure 2 above).
Appropriate histologic analysis with stains addressing proliferation, apoptosis, and
angiogenesis are performed. If one of the primary aims of the paper, however, is to show
that CHANGES in angiogenesis can be measured non-invasively with the flash
replenishment method, then the authors should include analyses at both pre- and post-
therapy time points.
Yes the reviewer is right, we measured the changes of different parameters of the tumor
non-invasively with the flash replenishment method pre- (baseline) and post- therapy
and compared the therapy group with the control group. Histologic analyses are
technically very difficult. For immunhistechemical analysis we would have had to
explant the tumor and then no further non-invasive measurements would have been
possible.
The title and the introduction refer to and discuss the effects of this therapy on invasion,
yet there are no measures of invasion included in the studies performed. As the
document stands, this claim is unfounded.
We changed the title and explained the connection between invasion, tumor growth,
proliferation and apoptose.
The concept of vascular normalization with anti-angiogenic therapy is ignored,yet such a
phenomenon may complicate the measurements proposed. This should be addressed.
On a more high-level view of this paper, there should be some comparison made as to
the efficacy of this treatment and the efficacy of other treatments currently used
clinically. Is this combination therapy more or less effective than others as tested in
murine or rat models? This could be accomplished simply by reference to published
material if not performed experimentally in a comparative fashion.
There are very few relevant manustricpts availabile that involve Hypopharynxtumor
cells in a rat or mouse model. We have added references to other published material and
comparisions with our results.
MINOR ESSENTIAL REVISIONS
The authors present international nonproprietary drug names as trade names. This
should be changed. Where the authors use the term Mesupron (which is a trade name),
this should be changed to the drug's INN, upamostat.
According to the reviewer request we changed all trade names into international drug
names.
Claims such as those made on lines 110, 116, and 128 should be stated as opinion or
substantiated with data, even if by reference. As an example, many targeted
therapeutics, when combined, can be highly toxic. Such blanket statements to not reflect
what is known in the field of targeted therapeutics as a whole.
Line 128 has been deleted.
All our remaining “claims” are substantiated by reference at the end of each sentence.
The treatment regimens should be clarified. Throughout the document, descriptions of
the actual treatments, including dosing regimens and the exact control methods are
imprecise and confusing. For example, does “0.1 ethanol” refer to 10% ethanol, 0.1 ml
ethanol, or something else? What is the concentration of the stock? If the formulations
have different vehicles, what is in the control? How much do they receive.
According to the reviewer request we amended the M&M passage to be more specific.
- 0.1 ml ethanol
- The control group received the same amount of solvents without any drug followed by
the Ulcogant for stomach protection.
On the boxplots, it is not clear whether some data points are treated as outliers (and
excluded from the statistical analysis), or just presented as outliers on the graph. If so,
there are a large number of outliers.
They are presented as outliers on the graph but were not excluded from the statistical
analysis.
Graphs need to be labelled with more complete legends and axis description so that they
may be more easily and quickly understood.
Legends for graphs should be written in scientific language to describe the data shown
in the graphs. Conclusions presented in the legends, if any, should be very limited in
length and scope.
According to the reviewer request we have edited the legends.
The discussion section should be written as more of a discussion and less as a
restatement of results, as it is currently written.
According to the reviewer request we have revised the discussion.
A few of the statements made reflect a poor understanding of the clinical care of these
patients, such as “histological follow-up of an anti-angiogenic therapy is clinically
impossible” and “in comparison to conventional treatment regimens such as
chemotherapy and radiotherapy, these forms have fewer side effects”. Consultation
should be made with a clinician prior to resubmission.
According to the reviewers request we have changed/deleted the formulation (6
clinicians are involved in this paper)
“Since a histological follow-up of an anti-angiogenic therapy is clinically infeasible, as it
is not appropriate for the patients to undergo repetitive biopsies within short time
spans, we choose the CEUS method to assess effects on tissue hemodynamics as a
surrogate for anti-angiogenic and invasion-inhibiting effects of the triple combination
therapy”.
Deleted: “in comparison to conventional treatment regimens such as chemotherapy and
radiotherapy, these forms have fewer side effects”:
Level of interest: An article whose findings are important to those with closely related
research interests
Quality of written English: Needs some language corrections before being published
Statistical review: No, the manuscript does not need to be seen by a statistician.
Declaration of competing interests: I declare that I have no competing interests.
Reviewer 3:
In this manuscript, the authors showed the triple therapy could lead to a significant
reduction of the number of vessels in the tumor compared to the placebo group. They
further demonstrated that the anti-angiogenic and invasion-inhibiting effects of the
triple therapy can be evaluated non-invasively with CEUS using the “flash
replenishment” method. These data in the paper are generally convincing.
There are several specific concerns that may limit enthusiasm for the manuscript as
written.
1. Did authors examine the effects of combinations of two therapies in these tumors
(e.g., WX-UK1 and Galardin, etc.)?
Yes , In a previous work, we analyzed single, double and triple therapy in an in vitro
model, (see answer above).
2. The triple therapy might be more toxic and have more side effects compared to the
single agents or in double combinations.
In our previous work, cellular metabolism was assessed in a standard MTT conversion
assay. Briefly, FaDu, HeLa, fibroblasts and HUVE cells (3 × 103 cells/well) were plated
in 96-well plates and analyzed at the time points indicated. No significant decrease or
increase in cell vitality was observed upon treatment of FaDu and HeLa cells with all
three compounds in comparison the single agents (see Zengel et al. BMC Cancer 2010
10:92 doi:10.1186/1471-2407-10-92.)
3. I suggest authors adding two or more time points for the follow-up measurements.
We are very sorry, but this is not possible, as the approved numbers of animal has
already been reached and further approval is not possible.
4. It would be more convincing if authors provide some representative CEUS images of
the tumors.
According to the reviewer request we have added some representative CEUS images
Baseline and follow up: Figure 6.
5. Since some inhibitors may produce off-target effects, have authors considered the
possibility of delivering silencing gene therapy to tumors using ultrasound-targeted
microbubble destruction?
This is a great idea. Unfortunately, we had no ultrasoundet-tergeted microbubble
availiable at the timepoint of our study. We only began to test different ultrasound-
targeted microbubbles a few months ago, well after the measurement timepoints of the
study.
Minor issues:
1. There are some redundancies in introduction/discussion section.
According to the reviewer request we have deleted the redundancies in the discussion
section.
2. In figure 4, the two lines should be clearly specified in the figure or in figure legend.
According to the reviewer request we have specified the two lines.
Thus, I would recommend to accept this manuscript after Minor Essential Revisions.
Level of interest: An article of importance in its field
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests: I declare that I have no competing interests.
