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Comments on "Sickle Erythrocytes Target Cytotoxics to Hypoxic Tumor Microvessels and Potentiate a Tumoricidal Response" in PloS ONE

Posted by fpruijn on 20 Jan 2013 at 23:08 GMT

This article describes a novel concept of using sickle erythrocytes (SSRBCs) to selectively target the hypoxic vascular microenvironment of solid tumours, which could be a new tool in the treatment of hypoxic solid tumours. However, a number of issues warrant comment.

In the Introduction it reads:

"Tirapazamine, the earliest prototype of this group demonstrated no survival benefit when added to standard chemotherapy and was associated with dose limiting myelosuppression related to activation of aerobic reductases in normal tissues [6]."

Possibly, the authors meant “… activation ""of tirapazamine"" by aerobic reductases in normal tissues.” This may be an explanation for the DLT but not one that appears to be supported by ref. [6]:

"Mild hypoxic conditions that occur in certain normal tissues, such as liver, retina and bone marrow, can activate TPZ and subsequently have effects on its toxicity profile [35-37]."

Those same authors of ref. [6] concluded:

"… several trial design flaws and significant treatment deviations may have led to the negative results in the Phase III trials reported."

Indeed, one paper subsequent to ref. [6] draws attention to this.(1)

In addition, although the primary target of tirapazamine is the hypoxic tumour cells anti-vascular effects have also been observed.(2)

Although the authors correctly state that to date no bioreductive prodrugs (or hypoxia-activated prodrugs; HAP) have been approved for clinical use it is fair to say that there are exciting times ahead for HAPs given that the HAP TH-302 is in advanced clinical trials through Threshold Pharmaceuticals and the molecularly-targeted HAP PR-610 (which releases pan-ErbB inhibitor that is effective against the T790M EGFR mutation that is frequently responsible for erlotinib resistance in NSCLC) is currently in phase I/II clinical trial through Proacta Inc.

The authors further write:

"Finally because of the heterogeneity in hypoxia between tumours of the same type, both groups require in vivo diagnostics to accurately measure hypoxia in order to select patients who can benefit most from these treatments [9]."

It is unclear why the authors seem to imply that their novel treatment method does not require pre-selection of patients with hypoxic tumours to achieve benefit particularly given that administration of SSRBCs needs to be combined with zinc protoporphyrin (ZnPP) alone or together with doxorubicin (ZnPP-D) to be efficacious. In addition, according to the proposed mechanism SSRBC-induced tumour vaso-occlusion occurs in hypoxic tumour vessels.

The authors do not explicitly state whether SSRBCs target the tumour vessels that are '"hypoxic'" or '"all'" tumour vessels. The impression is that it the target is the hypoxic vessels but it would help if the authors could elaborate on this and the experimental evidence; currently, it seems an inference. In other words, because SSRBCs selectively accumulate in tumour tissue and because tumours contain hypoxic regions SSRBCs target hypoxic tumour vessels. Although they conclude that there was no '"statistically significant'" accumulation of SSRBCs in normal tissues there was certainly a trend for SSRBC uptake in all tissues compared to NLRBCs (cf. Fig. 5). Therefore, the tentative conclusion may be that human SSRBCs selectively target murine (hypoxic?) tumour vessels relative to murine normal tissue vessels. The proposed mechanism is consistent with the experimental data but that does not constitute proof (in its broadest sense) that it is the correct mechanism.

Doxil is a pegylated liposomal form of doxorubicin. However, both are used interchangeably throughout the article; in Table S1 both “Dox” and “Doxil” are used and the authors should clarify whether they indeed used both forms and why; the Methods section only refers to doxorubicin (DOX).

Towards the end of the Introduction the authors refer to a “seminal report which precisely identified a central role for SSRBCs in targeting the upregulated/hypoxic tumor vasculature”. However, this ‘report’ turns out to be a patent and not a report in a peer-reviewed journal.

As a minor point, the name of the second author of ref. [8] should be corrected.

The tumour growth data (Kaplan-Meier) in Fig. 6 show the responses of the most effective combination treatments. It is obvious that the 4T1 murine tumour is an extremely fast growing tumour. It would have been nice to see the growth curves for the individual tumours as Supplementary Information particularly since that mice receiving SSRBC1× alone displayed significantly '"accelerated'" tumour growth compared to the PBS control.

In the Discussion the authors write:

"As a source of SSRBCs for clinical use, nucleated sickle progenitor cells (phenotypic and functional sickle cells) can be readily expanded/differentiated in vitro."

Since many readers (including this one) will be unfamiliar with this area it would have been helpful to support this with a reference.

As a forward-looking comment one would like to know how this potentially very useful tool performs in other murine tumours as well as human tumour xenograft models (although the vasculature and stroma will be host-derived), and how it may be implemented into clinical use.


1. Peters LJ, O'Sullivan B, Giralt J, et al.: Critical impact of radiotherapy protocol compliance and quality in the treatment of advanced head and neck cancer: Results from TROG 02.02. J Clin Oncol 28:2996-3001, 2010

2. Bains LJ, Baker JH, Kyle AH, et al.: Detecting vascular-targeting effects of the hypoxic cytotoxin tirapazamine in tumor xenografts using magnetic resonance imaging. Int J Radiat Oncol Biol Phys 74:957-965, 2009

Competing interests declared: I hold stock options in Proacta. I am an co-inventor of several patents on hypoxia-activated prodrugs as anti-cancer agents, in particular those covering SN 30000, which is an improved analogue of tirapazamine and which is currently in preclinical development by Cancer Research UK under a licence from the University of Auckland.

RE: Comments on "Sickle Erythrocytes Target Cytotoxics to Hypoxic Tumor Microvessels and Potentiate a Tumoricidal Response" in PloS ONE

DavidT replied to fpruijn on 25 Jan 2013 at 21:49 GMT

Response to Dr. Frederick Pruijn
We thank Dr. Pruijn for his comments and attention to our paper.
In Figure 1, hyperspectral imaging of the 4T1 tumor shows significant hypoxia in tumor blood vessels. At this time point, we also indicate that the 4T1 tumor shows diffuse pimonidazole uptake (see manuscript reference [46]: Vishwanath et. al., (2009) Neoplasia 11:889–900). Although not cited in our PLoS One paper, we have also compared hemoglobin saturation with phosphorescence lifetime imaging of oxygen sensitive nanoparticles in the 4T1 tumor, showing excellent correlation between the two (Palmer GM, Fontanella AN, Zhang G, Hanna G, Fraser CL, Dewhirst MW. Optical imaging of tumor hypoxia dynamics. Journal of Biomedical Optics. 2010;15(6):066021. doi: 10.1117/1.3523363). The combined data from hemoglobin saturation studies, oxygen nanoparticles and pimonidazole uptake supports our contention that both tumor vessels and tumor cells are hypoxic in the 4T1 tumor (see manuscript Discussion page e5343, right column last paragraph). In this hypoxic tumor mileau, Figures 3, 4 and corresponding text in Results plus the Supplemental video furnish qualitative and quantitative evidence that SSRBCs but not NLRBCs adhere to tumor microvessels, form micoraggregates and induce tumor vaso-occlusion. Figure 5 confirms these findings showing that SSRBC uptake in tumor tissue significantly exceeds that of normal RBCs whereas SSRBC and NLRBC uptake in three other organs is not significantly different. Collectively, these data provide compelling evidence that SSRBCs, but not NLRBCs, selectively adhere to hypoxic tumor blood vessels wherein they induce tumor vaso-occlusion.
Figure 6 illustrates the complete tumor growth delay study. The significantly accelerated growth of the SSRBC1x alone is indicated in the Table in Figure 6 and further highlighted in a full paragraph in the Discussion (page e5243, right column, last paragraph). In this paragraph, we indicate that this effect was reversible with the addition of ZnPP possibly related to acute hypoxia and HO-1 generation resulting from SSRBC-induced vaso-occlusion.
A “report” is defined as “…a written record or summary.” To this end, reference [31] provides a comprehensive written description of the use of sickle cells as a therapeutic agent against hypoxic solid tumors. The document, filed in 1999, was thoroughly examined for novelty/unobviousness over the prior art and fulfulled the rigorous PTO requirements for enablement and written description. The document further prevailed in an interference and was issued in 2010. This written record, therefore, provides the first tangible disclosure of SSRBCs as therapeutic agents against hypoxic tumors.
Abundant references to collection and processing of RBC progenitor cells may be found in the literature. Implementation of this treatment can take the form of ABO matched transfusions of sickle cells derived from in vitro expanded progenitors or autologous/allogeneic hematopoetic cell transplants using progenitor cells bioengineered to express the SS phenotype.
We are quite familiar with the expanded efforts to identify a “better” hypoxic cytotoxin and in fact, were involved in some of the seminal studies that led to the development of SN 30000 (Hicks KO, et al. Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs. Journal of the National Cancer Institute. 2006;98(16):1118-28. doi: 10.1093/jnci/djj306. PubMed PMID: 16912264.). We hold significant respect for the excellent work on novel hypoxic cytotoxins, as detailed by Dr. Pruijn in his letter. Certainly one would hope that one or more of these becomes accepted for treatment of hypoxic tumors. The most advanced of this newer class of hypoxic cytotoxins, TH 302, has been or is being studied extensively in phase I/II and III trials. However, it has yet to be approved for treatment of any disease. Tirapazamine also made it to phase III, where it failed for the reasons discussed above. Alternatively, there is no reason to exclude the eventual possibility that SSRBC therapy could be combined with hypoxic cytotoxins.
The correct name of the second author in reference [8] is Wilson WR.
Doxorubin, not Doxil was used in this study. The correction is duly noted.
David S. Terman
Mark W. Dewhirst

Competing interests declared: DST is sole inventor of US patent 7,803,637 filed August 30,
2000, issued September 28, 2010. All other authors have no competing interests