Elsevier

Cryobiology

Volume 61, Issue 3, December 2010, Pages 280-288
Cryobiology

Pre-conditioning cryosurgery: Cellular and molecular mechanisms and dynamics of TNF-α enhanced cryotherapy in an in vivo prostate cancer model system

https://doi.org/10.1016/j.cryobiol.2010.09.006Get rights and content

Abstract

Cryosurgery is increasingly being used to treat prostate cancer; however, a major limitation is local recurrence of disease within the previously frozen tissue. We have recently demonstrated that tumor necrosis factor alpha (TNF-α), given 4 h prior to cryosurgery can yield complete destruction of prostate cancer within a cryosurgical iceball. The present work continues the investigation of the cellular and molecular mechanisms and dynamics of TNF-α enhancement on cryosurgery. In vivo prostate tumor (LNCaP Pro 5) was grown in a dorsal skin fold chamber (DSFC) on a male nude mouse. Intravital imaging, thermography, and post-sacrifice histology and immunohistochemistry were used to assess iceball location and the ensuing biological effects after cryosurgery with and without TNF-α pre-treatment. Destruction was specifically measured by vascular stasis and by the size of histologic zones of injury (i.e., inflammatory infiltrate and necrosis). TNF-α induced vascular pre-conditioning events that peaked at 4 h and diminished over several days. Early events (4–24 h) include upregulation of inflammatory markers (nuclear factor-κB (NFκB) and vascular cell adhesion molecule-1 (VCAM)) and caspase activity in the tumor prior to cryosurgery. TNF-α pre-conditioning resulted in recruitment of an augmented inflammatory infiltrate at day 3 post treatment vs. cryosurgery alone. Finally, pre-conditioning yielded enhanced cryosurgical destruction up to the iceball edge at days 1 and 3 vs. cryosurgery alone. Thus, TNF-α pre-conditioning enhances cryosurgical lesions by vascular mechanisms that lead to tumor cell injury via promotion of inflammation and leukocyte (esp. neutrophil) recruitment.

Introduction

Prostate cryosurgery using intra-operative iceball imaging and modern Argon Joule–Thomson probe technology has increased markedly in the last decade [45], [47]. Although mostly accepted for salvage following local radiotherapy failure, it is now increasingly used as a primary treatment for locally advanced disease [29]. It is estimated that 6680 cryoablation procedures were performed in the United States in 2005 with 15,000 procedures projected in 2010 [13]. Five-year biochemical disease free status for cryosurgery is now comparable to published results for radiation and surgery in the prostate [27]. However, reproducible clinical application of cryosurgery continues to suffer from the inability of the technique to destroy cells at the periphery of the lesion (i.e., at the iceball edge) where the temperatures are above −40 °C [16]. Clearly, sub-lethal injury at the iceball edge may lead to cancer recurrence. However, over-freezing (creating an iceball beyond clinically apparent cancer) may result in damage of surrounding normal structures such as nerves and vessels, leading to complications [49]. As both under and over-freezing outcomes are undesirable, much research has focused on controlling destruction within the iceball by cellular, vascular and immunological mechanisms [4], [6], [17], [46]. It is now clear that one important approach to controlling disease at the edge of the prostate, where both the cancer and the iceball edge co-exist, is to augment these mechanisms with cryosurgical adjuvants as recently reviewed [19].

Several molecular adjuvants have been used to enhance cryosurgical injury within the iceball. These adjuvants can be broadly categorized into four groups: (1) thermophysical adjuvants to enhance the injury by ice crystal formation, (2) chemotherapeutic approaches to induce apoptosis (programmed cell death), (3) intravascular agents to induce vessel damage (and therefore ischemic necrosis), and (4) immunomodulators to stimulate immune-mediated tumor damage [19]. We have previously demonstrated that pre-treatment with TNF-α, a vascular agent, can completely destroy tumor throughout a cryosurgical iceball [20], [26].

TNF-α, isolated 30 years ago, is a multifunctional cytokine that plays a key role in apoptosis and cell survival as well as in inflammation and immunity. There are a number of mechanisms by which TNF-α can induce an anticancer effect, including: apoptosis [44], pro-coagulation and acute hemorrhagic necrosis [23], activation and mediation of macrophage and natural killer (NK) cell destruction [23], and occasionally the initiation of tumor specific immunity [3], [22], [37]. Our earlier results suggest that inflammation and vascular injury are critical specifically to TNF-α-mediated enhancement of cryosurgery [26]. However, further description of the timing and mechanisms of TNF-α pre-conditioning and enhancement of combinatorial treatment are needed for optimal translation to clinical use.

This work specifically investigates and reports on the time course and mechanism of TNF-α pre-treatment effects in tumors, and of combinatorial treatment (TNF-α pre-treatment + cryosurgery). While the systemic use of native TNF-α yields toxicity, we are able to topically apply it in the dorsal skin fold chamber without toxicity in this work. In other work we have also shown that safe systemic delivery is possible followed by cryosurgery with a gold nanoparticle carrier (CYT-6091) which may ultimately translate to clinical use [20]. Enhancement of cryosurgical injury in combinatorial treatment by histology is confirmed here as previously reported [4], [20], [25]. In addition, we show for the first time that enhancement is accompanied by the presence of a strong and sustained neutrophil inflammatory infiltrate. Also, new immunohistochemical results show that TNF-α-mediated enhancement correlates with translocation of NFκB, VCAM expression and upregulated expression of caspase 3 (a marker of apoptosis) in the cells and vasculature of the tumor.

Section snippets

Cell culture

LNCaP Pro 5 cells were transfected with plasmid DNA encoding the DsRed-express (Clontech, Mountain View, CA) fluorescent protein to permit monitoring of tumor growth in dorsal skin fold chambers (DSFCs) as described previously [20]. DsRed-LNCaP cells were cultured as adherent monolayers in Dulbecco’s modified Eagle’s medium (DMEM)/F12 media (BD Biosciences, San Jose, CA) supplemented with 10% of fetal bovine serum, antibiotics, and 10−9 mol/L dihydrotestosterone (DHT) as previously described [4].

Histological zones following cryosurgery without TNF-α

The maximum cryosurgical lesion without TNF-α pre-treatment presented at day 3. Cryoinjury was characterized by five histological zones extending radially outward from the probe location (Fig. 1). Zone 1: Immediately surrounding the cryoprobe a central necrotic zone was formed, characterized by intense eosinophilic staining of cells, loss of hematoxylin staining and loss of cellular detail. Blood vessels in this central necrotic zone were destroyed. In some specimens, scattered neutrophils were

Discussion

In recent years a new research thrust has emerged in cryosurgery that focuses on the use of molecular adjuvants to manipulate and enhance cellular and tissue cryodestruction [2], [6], [20], [41], [56]. The usage of these cryoadjuvants was recently reviewed [19]. For instance, as apoptosis is recognized as a mode of cell death at the periphery of the iceball, many studies used apoptosis inducers (chemotherapeutics) combined with cryosurgery at milder temperature (−40 to −0.5 °C) [5], [7], [14],

Acknowledgments

Institute for Engineering in Medicine, University of Minnesota; NIH RO1 NCl CA07528 for financial support. BioNet histology and digital imaging facilities are supported by NIH P30 CA77598 and P50 CA101955.

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