For HIPEC, synergistic effects of hyperthermia and doxorubicin are optimal when simultaneously combined

Dear Sir,

We thank Dr. Chen and colleagues for their interest in our published findings, Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia [1]. We have read their suggestions with interest. Below, we provide our response.

Clonogenic assay is the method of choice to quantify reproductive capacity in cells after exposure to ionizing radiation and other cytotoxic agents [2]. It has become an established assay to measure biological effects of hyperthermia, alone or in conjunction with other modalities, by providing a method to quantify cellular response with thermal dose (exposure time at elevated temperature) [2–7]. Given that it is an established tool to quantify the biological effects of hyperthermia and therapeutic enhancement with other modalities, it is the method of choice for the current study. Clonogenic assays integrate the culmination of multiple stress responses leading to loss of reproductive capacity in cells; however, they do not inform about specific pathway activation or mechanisms leading to reproductive cell death. Other assays measuring cell viability, metabolic function, membrane integrity, mechanisms of cell death, apoptosis, etc. are available and their use can provide insights into specific mechanisms; however, clonogenic assay is the only established method that provides a quantitative correlation with thermal dose and clinically relevant biological response. Thus, for quantitative comparisons of dose-response with hyperthermia and other modalities, the clonogenic assay remains the method of choice.

Hyperthermia and doxorubicin are pleiotropic cytotoxic agents affecting many cellular processes [5–36]. As such, they induce multiple (dose dependent) cell stress mechanisms, with cell death resulting from a failure of repair/response to ensure recovery and continued proliferation. Our quantitative comparison establishes that combining hyperthermia with doxorubicin leads to synergistic therapeutic effects in the three colorectal cancer cell lines studied. We note that two of these cell lines are human and the third is murine. Thus, our results demonstrate that this synergistic effect is not necessarily limited to human or murine cells. Further, we emphasize that such quantitative comparisons are essential to substantiate claims of synergistic effects. Whether this synergistic relationship exists across all known colorectal cancer cell lines, or cell lines representing other cancers remains an open question. We suggest that future studies begin with a similarly quantitative assessment of the dose-response relationship between hyperthermia and doxorubicin as a beginning point for detailed inquiry into biological mechanisms.

The conclusion that the observed synergism is not explained by increased intracellular doxorubicin levels was ascertained with only one cell line (HCT116), demonstrating that other mechanisms are responsible for the effect in this cell line. Despite the obvious limitation, the conclusion remains valid for this cell line. In the manuscript, we make no claim as to its prevalence or general applicability to other cell lines. We clearly state that further study with multiple cell lines is warranted; however, depending on individual phenotype and genotype, a range of responses can be expected vis a vis pleiotropic agents. Nevertheless, it motivates further examination to elucidate how this relates to other cells. The results present opportunities for further exploration to develop a deeper understanding of the effects of doxorubicin and heat.

We agree that further study is needed to ascertain mechanistic details giving rise to this synergism. For such studies, a careful selection of the appropriate assay(s) to probe individual stress responses is necessary. We suggest the careful selection of additional cell lines, having documented deficiencies in stress response and repair mechanisms to complement inquiry into the mechanistic details. We hope that our findings will motivate a renewed interest in research probing specific cell stress response pathways leading to a deeper understanding of hyperthermia + doxorubicin across multiple cell lines.

Concerning timing between heat and doxorubicin treatments, we recognize there is a broader implication for hyperthermia and doxorubicin combinations on which the authors of the letter focus; and, we are aware and agree that complex cellular and physiologic processes continue after heating. In this work, we focused on short time intervals because these are relevant for HIPEC, a clinical procedure used to treat various peritoneal neoplasms with heated (to hyperthermic temperature) chemotherapeutics, one of these being doxorubicin [37–43]. It was our intention to explore effects and time intervals relevant for this clinical procedure. Certainly, there is motivation to take a renewed interest in heat + doxorubicin combinations with varied timing that may be more relevant for other clinical applications. In their letter, Chen et al. mention several examples using modalities other than HIPEC. One of the mentioned applications is ablation of VX2 tumors. We note that ablation involves fundamentally different biological processes, and is a different clinical application [6]. Ablation was not the focus of our study [1]. It is, however, relevant for some modalities and we hope that results presented will prove informative for such future work.

In this work, we sought to identify whether there is a meaningful effect of sequence of treatment with heat + doxorubicin, and thermal dose in cellular response. In this context, our results provide information relevant for some clinical procedures, and we hope they will motivate others to reexamine the complex and potentially elegant interactions between these venerable agents.

Disclosure statement

No potential conflict of interest was reported by the author(s).