Comparison of ex vivo bioluminescence imaging, Alu-qPCR and histology for the quantification of spontaneous lung and bone metastases in subcutaneous xenograft mouse models.

Bioluminescence imaging (BLI) is a non-invasive state-of-the-art-method for longitudinal tracking of tumor cells in mice. The technique is commonly used to determine bone metastatic burden in vivo and also suitable ex vivo to detect even smallest bone micro-metastases in spontaneous metastasis xenograft models. However, it is unclear to which extent ex vivo BLI correlates with alternative methods for metastasis quantification. Here, we compared ex vivo BLI, human DNA-based Alu-qPCR, and histology for the quantification of bone vs. lung metastases, which are amongst the most common sites of metastasis in prostate cancer (PCa) patients and spontaneous PCa xenograft models. Data from 93 immunodeficient mice were considered, each of which were subcutaneously injected with luciferase/RGB-labeled human PCa PC-3 cells. The primary tumors were resected at ~ 0.75 cm³ and mice were sacrificed ~ 3 weeks after surgery and immediately examined by ex vivo BLI. Afterwards, the right lungs and hind limbs with the higher BLI signal (BLIHi bone) were processed for histology, whereas the left lung lobes and hind limbs with the lower BLI signal (BLILo bone) were prepared for Alu-qPCR. Our data demonstrate remarkable differences in the correlation coefficients of the different methods for lung metastasis detection (r ~ 0.8) vs. bone metastasis detection (r ~ 0.4). However, the BLI values of the BLIHi and BLILo bones correlated very strongly (r ~ 0.9), indicating that the method per se was reliable under identical limitations; the overall level of metastasis to contralateral bones was astonishingly similar. Instead, the level of lung metastasis only weakly to moderately correlated with the level of bone metastasis formation. Summarized, we observed a considerable discrepancy between ex vivo BLI and histology/Alu-qPCR in the quantification of bone metastases, which was not observed in the case of lung metastases. Future studies using ex vivo BLI for bone metastasis quantification should combine multiple methods to accurately determine metastatic load in bone samples.

Efficacy of zoledronic acid for the elimination of disseminated tumor cells in a clinically relevant, spontaneously metastatic prostate cancer xenograft model.

Bone metastases develop in >90 % of patients with castration-resistant prostate cancer (PCa) through complex interactions between the bone microenvironment and tumor cells. Previous androgen-deprivation therapy (ADT), which is known to cause bone loss, as well as anti-resorptive agents such as zoledronic acid (ZA), used to prevent skeletal complications, may influence these interactions and thereby the growth of disseminated tumor cells (DTC) in the bone marrow (BM). Here, a spontaneously metastatic xenograft tumor model of human PCa was further optimized to mimic the common clinical situation of ADT (castration) combined with primary tumor resection in vivo. The effects of these interventions, alone or in combination with ZA treatment, on tumor cell dissemination to the BM and other distant sites were analyzed. Metastatic burden was quantified by human-specific Alu-qPCR, bioluminescence imaging (BLI), and immunohistochemistry. Further, bone remodeling was assessed by static histomorphometry and serum parameters. Initial comparative analysis between NSG and SCID mice showed that spontaneous systemic dissemination of subcutaneous PC-3 xenograft tumors was considerably enhanced in NSG mice. Primary tumor resection and thereby prolonged observational periods resulted in a higher overall metastatic cell load at necropsy and tumor growth alone caused significant bone loss, which was further augmented by surgical castration. In addition, castrated mice showed a strong trend towards higher bone metastasis loads. Weekly treatment of mice with ZA completely prevented castration- and tumor-induced bone loss but had no effect on bone metastasis burden. Conversely, the total lung metastasis load as determined by BLI was significantly decreased upon ZA treatment. These findings provide a basis for future research on the role of ZA not only in preventing skeletal complications but also in reducing metastasis to other organs.

Development and Characterization of a Spontaneously Metastatic Patient-Derived Xenograft Model of Human Prostate Cancer.

Here we describe the establishment and characterization of an AR+, PSMA+, ERG+, PTEN-/-, CHD1+/- patient-derived xenograft (PDX) model termed 'C5', which has been developed from a 60 years old patient suffering from castration-resistant prostate cancer (CRPC). The patient underwent radical prostatectomy, showed early tumor marker PSA recurrence and, one year after surgery, abiraterone resistance. Subcutaneous C5 tumors can be serially transplanted between mice and grow within ~90 days to 1.5-2 cm³ tumors in SCID Balb/c mice (take rate 100%), NOD-scid IL2Rgnull (NSG) mice (100%) and C57BL/6 pfp-/-/rag2-/- mice (66%). In contrast, no tumor growth is observed in female mice. C5 tumors can be cryopreserved and show the same growth characteristics in vivo afterwards. C5 tumor cells do not grow stably in vitro, neither under two- nor three-dimensional cell culture conditions. Upon serial transplantation, some C5 tumors spontaneously disseminated to distant sites with an observable trend towards higher metastatic cell loads in scid compared to NSG mice. Lung metastases could be verified by histology by means of anti-PSMA immunohistochemistry, exclusively demonstrating single disseminated tumor cells (DTCs) and micro-metastases. Upon surgical resection of the primary tumors, such pulmonary foci rarely grew out to multi-cellular metastatic colonies despite doubled overall survival span. In the brain and bone marrow, the metastatic cell load present at surgery even disappeared during the post-surgical period. We provide shallow whole genome sequencing and whole exome sequencing data of C5 tumors demonstrating the copy number aberration/ mutation status of this PCa model and proving genomic stability over several passages. Moreover, we analyzed genomic and transcriptomic alterations during metastatic progression achieved by serial transplantation. This study describes a novel PCa PDX model that enables future research on several aspects of metastatic PCa, particularly for the AR+ , ERG+ , PTEN-/- PCa subtype.

Differential Proteome Analysis of Human Neuroblastoma Xenograft Primary Tumors and Matched Spontaneous Distant Metastases.

Metastasis formation is the major cause for cancer-related deaths and the underlying mechanisms remain poorly understood. In this study we describe spontaneous metastasis xenograft mouse models of human neuroblastoma used for unbiased identification of metastasis-related proteins by applying an infrared laser (IR) for sampling primary tumor and metastatic tissues, followed by mass spectrometric proteome analysis. IR aerosol samples were obtained from ovarian and liver metastases, which were indicated by bioluminescence imaging (BLI), and matched subcutaneous primary tumors. Corresponding histology proved the human origin of metastatic lesions. Ovarian metastases were commonly larger than liver metastases indicating differential outgrowth capacities. Among ~1,900 proteins identified at each of the three sites, 55 proteins were differentially regulated in ovarian metastases while 312 proteins were regulated in liver metastases. There was an overlap of 21 and 7 proteins up- and down-regulated at both metastatic sites, respectively, most of which were so far not related to metastasis such as LYPLA2, EIF4B, DPY30, LGALS7, PRPH, and NEFM. Moreover, we established in vitro sublines from primary tumor and metastases and demonstrate differences in cellular protrusions, migratory/invasive potential and glycosylation. Summarized, this work identified several novel putative drivers of metastasis formation that are tempting candidates for future functional studies.

Picosecond Infrared Laser (PIRL) Application in Stapes Surgery-First Experience in Human Temporal Bones.

OBJECTIVE: Using a contact-free laser technique for stapedotomy reduces the risk of mechanical damage of the stapes footplate. However, the risk of inner ear dysfunction due to thermal, acoustic, or direct damage has still not been solved. The objective of this study was to describe the first experiences in footplate perforation in cadaver tissue performed by the novel Picosecond-Infrared-Laser (PIRL), allowing a tissue preserving ablation. PATIENTS AND INTERVENTION: Three human cadaver stapes were perforated using a fiber-coupled PIRL. The results were compared with footplate perforations performed with clinically applied Er:YAG laser. Therefore, two different laser energies for the Er:YAG laser (30 and 60 mJ) were used for footplate perforation of three human cadaver stapes each. MAIN OUTCOME MEASURE: Comparisons were made using histology and environmental scanning electron microscopy (ESEM) analysis. RESULTS: The perforations performed by the PIRL (total energy: 640-1070 mJ) revealed a precise cutting edge with an intact trabecular bone structure and no considerable signs of coagulation. Using the Er:YAG-Laser with a pulse energy of 30 mJ (total energy: 450-600 mJ), a perforation only in the center of the ablation zone was possible, whereas with a pulse energy of 60 mJ (total energy: of 195-260 mJ) the whole ablation zone was perforated. For both energies, the cutting edge appeared irregular with trabecular structure of the bone only be conjecturable and signs of superficial carbonization. CONCLUSION: The microscopic results following stapes footplate perforation suggest a superiority of the PIRL in comparison to the Er:YAG laser regarding the precision and tissue preserving ablation.

Comparative study of wound healing in rat skin following incision with a novel picosecond infrared laser (PIRL) and different surgical modalities.

BACKGROUND AND OBJECTIVE: As a result of wound healing the original tissue is replaced by dysfunctional scar tissue. Reduced tissue damage during surgical procedures beneficially affects the size of the resulting scar and overall healing time. Thus the choice of a particular surgical instrument can have a significant influence on the postoperative wound healing. To overcome these problems of wound healing we applied a novel picosecond infrared laser (PIRL) system to surgical incisions. Previous studies indicated that negligible thermal, acoustic, or ionization stress effects to the surrounding tissue results in a superior wound healing. STUDY DESIGN/MATERIALS AND METHODS: Using the PIRL system as a surgical scalpel, we performed a prospective wound healing study on rat skin and assessed its final impact on scar formation compared to the electrosurgical device and cold steel. As for the incisions, 6 full-thickness, 1-cm long-linear skin wounds were created on the dorsum of four rats using the PIRL, an electrosurgical device, and a conventional surgical scalpel, respectively. Rats were euthanized after 21 days of wound healing. The thickness of the subepithelial fibrosis, the depth and the transverse section of the total scar area of each wound were analyzed histologically. RESULTS: After 21 days of wound healing the incisions made by PIRL showed minor scar tissue formation as compared to the electrosurgical device and the scalpel. Highly significant differences (P < 0.001) were noted by comparing the electrosurgical device with PIRL and scalpel. The transverse section of the scar area also showed significant differences (P = 0.043) when comparing PIRL (mean: 141.46 mm2; 95% CI: 105.8-189.0 mm2) with scalpel incisions (mean: 206.82 mm2; 95% CI: 154.8-276.32 mm2). The subepithelial width of the scars that resulted from using the scalpel were 1.3 times larger than those obtained by using the PIRL (95% CI: 1.0-1.6) though the difference was not significant (P < 0.083). CONCLUSIONS: The hypothesis that PIRL results in minimal scar formation with improved cosmetic outcomes was positively verified. In particular the resection of skin tumors or pathological scars, such as hypertrophic scars or keloids, are promising future fields of PIRL application.