Local radiotherapy reduces circulating tumor cells and metastatic progression : Role of circulating cell detection in response to vascular damaging therapies

Noninvasive biological readouts of tumor metastatic risk and therapeutic efficacy are needed as healthcare costs rise. Conventional imaging modalities are expensive; require significant periods of time to see appreciable change and repetitive use increases risk of hazardous side effects. Utilizing circulating tumor cells (CTCs) as a minimally invasive biological readout of tumor progression or therapeutic response is increasingly being studied. While focus has primarily been on the predictive value of naturally occurring CTCs, a more recent effort has emerged regarding the potential consequence of therapy induced CTCs. Here we report an investigation on the acute and long-term effect of vascular disrupting therapies (high-dose radiotherapy and tumor necrosis factor-alpha (TNF)) on CTCs monitored with a non-invasive real-time system. Acute mobilization of CTCs into the blood following both radiation and TNF treatment was observed. There was no increase in metastasis frequency or extent between control and TNF-treated mice; however, a significant reduction in lung metastasis was noted in the radiotherapy alone group. Mice treated with both TNF and radiotherapy had a slightly elevated metastatic profile between that of radiation alone and control (untreated) tumors. Possible mechanisms based on therapy specific vessel disruption and cell death are discussed. Overall, CTCs correlated with tumor progression and suggest CTC enumeration described herein may be useful in clinical management of solid tumor malignancies. M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Introduction Therapy associated disruption of tumor vasculature allows immune cellular transit from blood into tumor 1,2 as well as creates a possibility for cells to exit from tumor into blood. The presence of breast tumor derived cells in blood have been reported to increase the risk of metastasis 3 leading to CTC-based tests becoming more accepted in oncological practice . However, little work was done to analyze the risks of tumor metastatic progression stemming from therapy induced vascular disruption . Fractionated radiation was recently reported to augment the release of viable CTCs into circulation potentially retaining metastatic potential 6,7 in patients with non-small cell lung cancer and squamous cell carcinoma of the head and neck. Routine biopsy and pressure also have been reported to temporarily increase the number of tumor cells in circulation as a result of tumor vessel damage . Thus, for the success of a multistage anti-cancer therapy based on disruption of tumor vasculature, it is crucial to understand both shortand long-term effects of vascular damage on release of CTCs and any possible influence on extent of metastatic disease. The majority of patients with unresectable or metastatic solid tumors receive some type of radiotherapy during the course of treatment and, stereotactic body radiotherapy (highdose radiotherapy) is increasingly being used in radiation oncology practice . The higher doses of radiation accompanying this treatment are being deposited within tumor tissues and have led to observations of associated tumor vessel damage , and release of viable circulating tumor cells (CTCs) . The vascular effects of high-dose radiotherapy are not completely understood or appreciated ; moreover, a combination of vascular-disrupting and radiation-based therapies increasingly attracts attention 13 by providing a possibility of targeting both well oxygenated and hypoxic (low oxygen) tumor regions. We recently confirmed that a combination of vascular disrupting TNF-based nanoparticles, CYT-6091, with hypofractionated radiation induces synergistic tumor growth delay . The biological effects (vascular hemorrhaging) of TNF were M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT concentrated in radioresistant, hypoxic regions acting complementary to radiation therapy which has lower efficacy in such regions. Herewith, we hypothesized that vascular damaging high-dose radiation and/or CYT6091 may lead to a measurable increase in CTCs detectable by our in vivo fluorescence detection platform. Furthermore, we sought to assess the possible use of CTC indices as markers of primary tumor treatment efficacy and whether or not certain vascular damaging therapies had distinguishable effects on CTC levels or correlated to metastatic progression. An in vivo flow cytometry method for real time CTC quantification developed by our group 10,15 provided an opportunity to reveal short term (within minutes) as well as enumerate at later dates a snapshot of CTCs after radiation or anti-vascular treatment. Prior to initiating the study described herein, an initial study was conducted to ascertain if CTCs are released following CYT-6091 therapy in a translational model. Syngeneic 4T1 murine breast tumors expressing GFP and grown in balb/c mice indicated a 3-fold increase in CTCs in a time window in-line with the known vascular damaging effects of CYT-6091 (data not shown). M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT Materials and Methods Principles of in vivo fluorescence flow cytometry (FFC) detection Real-time quantification of CTCs in mouse blood was performed by means of in vivo single color fluorescence flow cytometry described elsewhere . A 488-nm diode laser (total power of 7 mW in the sample) was used to excite fluorescent CTCs 17 circulating in arterial vessels. A 40x objective (NA 0.75, Plan Fluor, Nikon) was used to focus the laser beam into an arterial vessel and laser light and fluorescence were separated using dichroic mirrors. A cylindrical lens (focal length of 250 mm) shaped the laser beam in the sample into a line (50×5 μm). The fluorescence was filtered with a bandpass filter 520±15 nm (Semrock, Inc., Rochester, NY) and detected by a photomultiplier (PMT) tube (PMT, R928, Hamamatsu, Co., Bridgewater, NJ). The PMT signal was acquired using a PCI-5124 digitizer (National Instruments, Austin, TX) and analyzed using custom MatLab software (MathWorks, Natick, MA). Cell lines and tumor model Mouse mammary carcinoma, 4T1, cells transfected with green fluorescent protein (4T1-GFP) were cultured at 37○C and 5% CO2 in DMEM 4.5 g/L D-glucose (Mediatech Inc., Manassas, VA) supplemented with 10% FBS. For tumor inoculation, cells growing exponentially were harvested at 80% confluence with 0.125% trypsin, suspended in serum free media at 5 x 10 cells/0.05 mLs, and 0.05 mLs implanted subcutaneously in the left rear limb of female Nu/Nu mice (Harlan; weighing 20-25 g). Microchip transponders implanted in the flank were used for mouse identification (Bio Medic Data Systems, Inc., Seaford, DE). The 4T1 breast cancer model was chosen because it is a well-established model of metastatic cancer (e.g. sites of metastasis in lymph node and lung). Implantation in immune compromised, Nu/Nu mice, was inline with previously optimized conditions for monitoring CTCs . All animal procedures M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT were performed with approval by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA). Dorsal skinfold window chamber model and monitoring vascular permeability The dorsal skinfold window chamber (DSWC) model was prepared as described previously . Briefly, custom aluminum frames with an approximately 12 mm diameter window opening were implanted on the dorsal skinfold. Tissue was removed from one side of the chamber leaving the subcutaneous tissue of the opposite side exposed for tumor spheroid implantation. 4T1-GFP cells grown as spheroids using the hanging drop method were implanted and a glass coverslip sealed the exposed tissue. Animals were anesthetized with 1% isoflurane and placed in a custom holder for window chamber visualization on an Olypmus IX71 inverted microscope. Tumor tissue and neovascularization was confirmed by presence of GFP with characteristic tumor vessels observed throughout the fluorescent tissue. Mice were treated with sterile H2O (100 uL, n=3) or CYT-6091 (250 μg/kg, n = 3) IV via the lateral tail vein 1 hour prior to intracardiac injection of 0.8 mg 70kD texas red labeled dextrans (Invitrogen) for imaging of drug-induced vascular permeability. Time-lapse images were collected at 5, 20 and 35 minutes post dextran injection. Treatments and tumor growth response Rear limb tumors were grown to an average size of 190 mm and randomized to various treatment groups: tumor control without treatment (n=6), CYT-6091 (250 μg/kg q2dx2; n=6), radiation (12 Gy q2dx3; n=6), and combined therapy (n=6). Ionizing radiation was administered using a Faxitron X-ray cabinet system (CP-160, Faxitron X-Ray Corp., Wheeling, IL) at a dose rate of 1.079 Gy/min (150 kVp and 6.6 mA) under ketamine/xylazine anesthesia. TNF was delivered via a pegylated gold nanoparticle carrier , administered IV on day 0 and day 2 M AN US CR IP T AC CE PT ED ACCEPTED MANUSCRIPT immediately following the first two rounds of radiation for the combined treatment group. Tumor diameter was measured manually in the xand y-axis using calipers. The tumor volume was calculated as: 2 ) ( diameter short diameter long Volume × = , and plotted as average tumor