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.

REFERENCES

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 http://dx.doi.org/10.1200...

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 http://dx.doi.org/10.1016...