Altering calcium influx for selective destruction of breast tumor.

BACKGROUND: Human triple-negative breast cancer has limited therapeutic choices. Breast tumor cells have depolarized plasma membrane potential. Using this unique electrical property, we aim to develop an effective selective killing of triple-negative breast cancer. METHODS: We used an engineered L-type voltage-gated calcium channel (Cec), activated by membrane depolarization without inactivation, to induce excessive calcium influx in breast tumor cells. Patch clamp and flow cytometry were used in testing the killing selectivity and efficiency of human breast tumor cells in vitro. Bioluminescence and ultrasound imaging were used in studies of human triple-negative breast cancer cell MDA-MB-231 xenograft in mice. Histological staining, immunoblotting and immunohistochemistry were used to investigate mechanism that mediates Cec-induced cell death. RESULTS: Activating Cec channels expressed in human breast cancer MCF7 cells produced enormous calcium influx at depolarized membrane. Activating the wild-type Cav1.2 channels expressed in MCF7 cells also produced a large calcium influx at depolarized membrane, but this calcium influx was diminished at the sustained membrane depolarization due to channel inactivation. MCF7 cells expressing Cec died when the membrane potential was held at -10 mV for 1 hr, while non-Cec-expressing MCF7 cells were alive. MCF7 cell death was 8-fold higher in Cec-expressing cells than in non-Cec-expressing cells. Direct injection of lentivirus containing Cec into MDA-MB-231 xenograft in mice inhibited tumor growth. Activated caspase-3 protein was detected only in MDA-MB-231 cells expressing Cec, along with a significantly increased expression of activated caspase-3 in xenograft tumor treated with Cec. CONCLUSIONS: We demonstrated a novel strategy to induce constant calcium influx that selectively kills human triple-negative breast tumor cells.

Third Prize: Comparison of radical nephrectomy, laparoscopic microwave thermotherapy, cryotherapy, and radiofrequency ablation for destruction of experimental VX-2 renal tumors in rabbits.

BACKGROUND AND PURPOSE: Currently available minimally invasive renal tumor-ablation procedures include cryotherapy, radiofrequency ablation, and microwave thermotherapy. In this study, we investigated the ability of these three approaches to destroy experimental renal tumors in rabbits. The mechanism of potential tumor metastasis was also explored. MATERIALS AND METHODS: The VX-2 tumor line is an aggressive rabbit epidermoid tumor with a high metastatic potential. An initial experiment comparing cooled-tip microwave thermotherapy with cryotherapy and radical nephrectomy for treatment of small VX-2 tumors revealed that all microwave-treated rabbits had local recurrence and that several also had diffuse intraperitoneal carcinomatosis. In view of these results, a second experiment was performed in which 45 New Zealand White rabbits were implanted laparoscopically with VX-2 xenografts underneath the kidney capsule and divided into five groups of 9 each. The test groups were microwave thermotherapy with a 3.5-mm cooled-tip probe, microwave thermotherapy with a 3.5-mm noncooled- tip probe, radiofrequency ablation with a 1.5-mm cooled-tip probe, radiofrequency ablation with a 1.5- mm non-cooled tip probe, and cryotherapy with a 2.3-mm cryoprobe. The control groups were five rabbits that were not treated, five rabbits with tumors that had the tumor pierced with a probe but were untreated, and five rabbits that underwent nephrectomy after piercing of the tumor. Treatment was initiated 5 days after tumor implantation. One month later, all animals were euthanized and autopsied. RESULTS: At 5 days after tumor implantation, laparoscopic inspection revealed no visible peritoneal metastases. At 1 month, in the cooled and non-cooled microwave-thermotherapy groups, carcinomatosis occurred in five and six of nine animals, respectively. In comparison, carcinomatosis was detected in two of nine animals in the cryotherapy group at autopsy. With respect to cooled and non-cooled radiofrequency ablation, carcinomatosis was observed in four of nine rabbits in each group. In the control groups, none of the animals with unpierced tumors exhibited carcinomatosis, while carcinomatosis was seen in two of the five rabbits with tumor violated by piercing and in three of the five rabbits that underwent immediate nephrectomy after piercing of the tumor. CONCLUSION: Carcinomatosis occurred most frequently in animals treated with microwave thermotherapy, followed by radiofrequency ablation, and lastly cryoablation. The simple act of piercing a highly aggressive tumor can result in local spread. More disconcerting, and less well understood, is why certain ablative modalities appear to increase the rate of intraperitoneal spread.

Cryothermic and hyperthermic treatments of human leiomyomata and adjacent myometrium and their implications for laparoscopic surgery.

STUDY OBJECTIVE: To evaluate the effects and feasibility of direct cryothermic and hyperthermic therapy on leiomyomata and adjacent myometrium, and to contribute to evidence-based treatment thresholds based on measurements of direct cell injury. DESIGN: Experimental study (Canadian Task Force classification II-2). SETTING: University hospital. SUBJECTS: Leiomyoma and myometrium tissue from 10 women undergoing total abdominal hysterectomy with or without bilateral salpingo-oophorectomy. INTERVENTION: In vitro cryothermic or hyperthermic therapy was performed with representative leiomyoma and myometrium tissue samples. Using a directional solidification stage to simulate cryothermic therapy, 10 leiomyoma and 6 myometrium specimens were cooled in vitro at a rate of -5 degrees C/minute to end temperatures of -20 degrees, -40 degrees, -60 degrees, and -80 degrees C with a 15-minute hold period and then rapidly thawed to 21 degrees C. Hyperthermic therapy was simulated using a preheated 45 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, and 80 degrees C constant temperature copper heating block with a 10-minute treatment period. In conjunction with tissue culturing and control tissues, cell death was assessed with routine histology and viability dyes (ethidium homodimer/Hoechst). MEASUREMENTS AND MAIN RESULTS: In cryothermic results, leiomyomata cell death (LCD) increased from 12% to 27% by histology and 26% to 38% by viability dye assay over the thermal range from -20 degrees to -80 degrees C, respectively. Myometrial cell death (MCD) increased from 10% to 12% and 4% to 20% for the same measurements, respectively. Whereas MCD appeared relatively stable from -40 degrees to -80 degrees C, it was significantly less than LCD over this range (p <0.05). For hyperthermic results, LCD increased from 17% to 88% by histology with progressive temperature increase from 45 degrees to 80 degrees C, respectively. The MCD showed a similar increase from 16% to 91% by histology over this temperature range. Hyperthermic histology and dye assay results were similar for LCD and MCD. CONCLUSIONS: In comparison with myometrium, leiomyomata showed greater direct cryothermic and equal hyperthermic cell injury. Whereas cell death increased up to 70 degrees C and down to -80 degrees C, the interval increases in cell injury diminished with more extreme temperatures. In vivo studies of combined direct and ischemic vascular injury thresholds have yet to be performed, but direct LCD matrixes determined in this study will help provide guidelines for minimally invasive surgical techniques for the treatment of leiomyomata.