Monitoring Early Response to Anti-Angiogenic Therapy: Diffusion-Weighted Magnetic Resonance Imaging and Volume Measurements in Colon Carcinoma Xenografts

Objectives: To evaluate the use of diffusion-weighted MRI (DW-MRI) and volume measurements for early monitoring of antiangiogenic therapy in an experimental tumor model. Materials and Methods: 23 athymic nude rats, bearing human colon carcinoma xenografts (HT-29) were examined before and after 6 days of treatment with regorafenib (n=12) or placebo (n=11) in a clinical 3-Tesla MRI. For DW-MRI, a single-shot EPI sequence with 9 b-values (10-800 s/mm(2)) was used. The apparent diffusion coefficient (ADC) was calculated voxelwise and its median value over a region of interest, covering the entire tumor, was defined as the tumor ADC. Tumor volume was determined using T2-weighted images. ADC and volume changes between first and second measurement were evaluated as classifiers by a receiver-operator-characteristic (ROC) analysis individually and combined using Fisher's linear discriminant analysis (FLDA). Results: All ADCs and volumes are stated as median +/- standard deviation. Tumor ADC increased significantly in the therapy group (0.76 +/- 0.09x10(-3) mm(2)/s to 0.90 +/- 0.12x10(-3) mm(2)/s;p<0.001), with significantly higher changes of tumor ADC than in the control group (0.10 +/- 0.11x10(-3) mm(2)/s vs. 0.03 +/- 0.09x10(-3) mm(2)/s;p = 0.027). Tumor volume increased significantly in both groups (therapy: 347.8 +/- 449.1 to 405.3 +/- 823.6 mm(3);p = 0.034;control: 219.7 +/- 79.5 to 443.7 +/- 141.5 mm(3);p<0.001), however, the therapy group showed significantly reduced tumor growth (33.30 +/- 47.30% vs. 96.43 +/- 31.66%;p<0.001). Area under the curve and accuracy of the ADC-based ROC analysis were 0.773 and 78.3%;and for the volume change 0.886 and 82.6%. The FLDA approach yielded an AUC of 0.985 and an accuracy of 95.7%. Conclusions: Regorafenib therapy significantly increased tumor ADC after 6 days of treatment and also significantly reduced tumor growth. However, ROC analyses using each parameter individually revealed a lack of accuracy in discriminating between therapy and control group. The combination of both parameters using FLDA substantially improved diagnostic accuracy, thus highlighting the potential of multi-parameter MRI as an imaging biomarker for non-invasive early tumor therapy monitoring

Monitoring Cell Death in Regorafenib-Treated Experimental Colon Carcinomas Using Annexin-Based Optical Fluorescence Imaging Validated by Perfusion MRI

Objective To investigate annexin-based optical fluorescence imaging (OI) for monitoring regorafenib-induced early cell death in experimental colon carcinomas in rats, validated by perfusion MRI and multiparametric immunohistochemistry. Materials and Methods Subcutaneous human colon carcinomas (HT-29) in athymic rats (n = 16) were imaged before and after a one-week therapy with regorafenib (n = 8) or placebo (n = 8) using annexin-based OI and perfusion MRI at 3 Tesla. Optical signal-to-noise ratio (SNR) and MRI tumor perfusion parameters (plasma flow PF, mL/100mL/min;plasma volume PV,%) were assessed. On day 7, tumors underwent immunohistochemical analysis for tumor cell apoptosis (TUNEL),proliferation (Ki-67),and microvascular density (CD31). Results Apoptosis-targeted OI demonstrated a tumor-specific probe accumulation with a significant increase of tumor SNR under therapy (mean Delta +7.78 +/- 2.95, control: -0.80 +/- 2.48, p = 0.021). MRI detected a significant reduction of tumor perfusion in the therapy group (mean Delta PF -8.17 +/- 2.32 mL/100 mL/min, control -0.11 +/- 3.36 mL/100 mL/min, p = 0.036). Immunohistochemistry showed significantly more apoptosis (TUNEL;11392 +/- 1486 vs. 2921 +/- 334, p = 0.001),significantly less proliferation (Ki-67;1754 +/- 184 vs. 2883 +/- 323, p = 0.012),and significantly lower microvascular density (CD31;107 +/- 10 vs. 182 +/- 22, p = 0.006) in the therapy group. Conclusions Annexin-based OI allowed for the non-invasive monitoring of regorafenib-induced early cell death in experimental colon carcinomas, validated by perfusion MRI and multiparametric immunohistochemistry

Correlation of Perfusion MRI and F-18-FDG PET Imaging Biomarkers for Monitoring Regorafenib Therapy in Experimental Colon Carcinomas with Immunohistochemical Validation

Objectives To investigate a multimodal, multiparametric perfusion MRI/F-18-fluoro-deoxyglucose (F-18-FDG)-PET imaging protocol for monitoring regorafenib therapy effects on experimental colorectal adenocarcinomas in rats with immunohistochemical validation. Materials and Methods Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n = 17 (n = 10 therapy group;n = 7 control group) female athymic nude rats (Hsd: RH-Foxn1(mu)). Animals were imaged at baseline and after a one-week daily treatment protocol with regorafenib (10 mg/kg bodyweight) using a multimodal, multiparametric perfusion MRI/F-18-FDG-PET imaging protocol. In perfusion MRI, quantitative parameters of plasma flow (PF, mL/100 mL/min), plasma volume (PV,%) and endothelial permeability-surface area product (PS, mL/100 mL/min) were calculated. In F-18-FDG-PET, tumor-to-background-ratio (TTB) was calculated. Perfusion MRI parameters were correlated with TTB and immunohistochemical assessments of tumor microvascular density (CD-31) and cell proliferation (Ki-67). Results Regorafenib significantly (p<0.01) suppressed PF (81.1 +/- 7.5 to 50.6 +/- 16.0 mL/100mL/min), PV (12.1 +/- 3.6 to 7.5 +/- 1.6%) and PS (13.6 +/- 3.2 to 7.9 +/- 2.3 mL/100mL/min) as well as TTB (3.4 +/- 0.6 to 1.9 +/- 1.1) between baseline and day 7. Immunohistochemistry revealed significantly (p<0.03) lower tumor microvascular density (CD-31, 7.0 +/- 2.4 vs. 16.1 +/- 5.9) and tumor cell proliferation (Ki-67, 434.0 +/- 62.9 vs. 663.0 +/- 98.3) in the therapy group. Perfusion MRI parameters Delta PF, Delta PV and Delta PS showed strong and significant (r = 0.67-0.78;p<0.01) correlations to the PET parameter Delta TTB and significant correlations (r = 0.57-0.67;p<0.03) to immunohistochemical Ki-67 as well as to CD-31-stainings (r = 0.49-0.55;p<0.05). Conclusions A multimodal, multiparametric perfusion MRI/PET imaging protocol allowed for non-invasive monitoring of regorafenib therapy effects on experimental colorectal adenocarcinomas in vivo with significant correlations between perfusion MRI parameters and F-18-FDG-PET validated by immunohistochemistry

Contrast-Enhanced Ultrasound with VEGFR2-Targeted Microbubbles for Monitoring Regorafenib Therapy Effects in Experimental Colorectal Adenocarcinomas in Rats with DCE-MRI and Immunohistochemical Validation

Objectives To investigate contrast-enhanced ultrasound (CEUS) with VEGFR2-targeted microbubbles for monitoring therapy effects of regorafenib on experimental colon carcinomas in rats with correlation to dynamic contrast-enhanced MRI (DCE-MRI) and immunohistochemistry. Materials and Methods: Human colorectal adenocarcinoma xenografts (HT-29) were implanted subcutaneously in n =21 (n = 11 therapy group;n = 10 control group) female athymic nude rats (Hsd: RH-Foxn1 (mu)). Animals were imaged at baseline and after a one-week daily treatment with regorafenib or a placebo (10 mg/kg bodyweight), using CEUS with VEGFR2-targeted microbubbles and DCE-MRI. In CEUS tumor perfusion was assessed during an early vascular phase (wash-in area under the curve = WiAUC) and VEGFR2-specific binding during a late molecular phase (signal intensity after 8 (SI8min) and 10 minutes (SI10min)), using a conventional 15L8 linear transducer (transmit frequency 7 MHz, dynamic range 80 dB, depth 25 mm). In DCE-MRI functional parameters plasma flow (PF) and plasma volume (PV) were quantified. For validation purposes, CEUS parameters were correlated with DCE-MRI parameters and immunohistochemical VEGFR2, CD31, Ki-67 and TUNEL stainings. Results: CEUS perfusion parameter WiAUC decreased significantly (116,989 +/- 77,048 a.u. to 30,076 +/- 27,095a.u.;p = 0.005) under therapy with no significant changes (133,932 +/- 65,960 a.u. to 84,316 +/- 74,144 a.u.;p = 0.093) in the control group. In the therapy group, the amount of bound microbubbles in the late phase was significantly lower in the therapy than in the control group on day 7 (SI8min: 283 +/- 191 vs. 802 +/- 460 a.u.;p = 0.006);SI10min: 226 +/- 149 vs. 645 +/- 461 a.u.;p = 0.009). PF and PV decreased significantly (PF: 147 +/- 58 mL/100 mL/min to 71 +/- 15 mL/100 mL/min;p = 0.003;PV: 13 +/- 3% to 9 +/- 4%;p = 0.040) in the therapy group. Immunohistochemistry revealed significantly fewer VEGFR2 (7.2 +/- 1.8 vs. 17.8 +/- 4.6;p < 0.001), CD31 (8.1 +/- 3.0 vs. 20.8 +/- 5.7;p < 0.001) and Ki-67 (318.7 +/- 94.0 vs. 468.0 +/- 133.8;p = 0.004) and significantly more TUNEL (672.7 +/- 194.0 vs. 357.6 +/- 192.0;p = 0.003) positive cells in the therapy group. CEUS parameters showed significant (p < 0.05) correlations to DCE-MRI parameters and immunohistochemistry. Conclusions CEUS with VEGFR2-targeted microbubbles allowed for monitoring regorafenib functional and molecular therapy effects on experimental colorectal adenocarcinomas with a significant decline of CEUS and DCE-MRI perfusion parameters as well as a significant reduction of specifically bound microbubbles under therapy, consistent with a reduced expression of VEGFR2

18F–FDG-PET/CT and diffusion-weighted MRI for monitoring a BRAF and CDK 4/6 inhibitor combination therapy in a murine model of human melanoma

Background: The purpose of the study was to investigate a novel BRAF and CDK 4/6 inhibitor combination therapy in a murine model of BRAF-V600-mutant human melanoma monitored by F-18-FDG-PET/CT and diffusionweighted MRI (DW-MRI). Methods: Human BRAF-V600-mutant melanoma (A375) xenograft-bearing balb/c nude mice (n = 21) were imaged by 18F-FDG-PET/CT and DW-MRI before (day 0) and after (day 7) a 1-week BRAF and CDK 4/6 inhibitor combination therapy (n = 12;dabrafenib, 20 mg/kg/d;ribociclib, 100 mg/kg/d) or placebo (n = 9). Animals were scanned on a small animal PET after intravenous administration of 20 MBq F-18-FDG. Tumor glucose uptake was calculated as the tumor-to-liver-ratio (TTL). Unenhanced CT data sets were subsequently acquired for anatomic coregistration. Tumor diffusivity was assessed by DW-MRI using the apparent diffusion coefficient (ADC). Anti-tumor therapy effects were assessed by ex vivo immunohistochemistry for validation purposes (microvascular density -CD31;tumor cell proliferation -Ki-67). Results: Tumor glucose uptake was significantly suppressed under therapy (Delta TTLTherapy -1.00 +/- 0.53 vs.Delta TTLControl 0.85 +/- 1.21;p < 0.001). In addition, tumor diffusivity was significantly elevated following the BRAF and CDK 4/6 inhibitor combination therapy (Delta ADC(Therapy) 0.12 +/- 0.14 x 10(-3) mm(2)/s;Delta ADCControl -0.12 +/- 0.06 x 10(-3) mm(2)/s;p < 0.001). Immunohistochemistry revealed a significant suppression of microvascular density (CD31, 147 +/- 48 vs. 287 +/- 92;p = 0.001) and proliferation (Ki-67, 3718 +/- 998 vs. 5389 +/- 1332;p = 0.007) in the therapy compared to the control group. Conclusion: A novel BRAF and CDK 4/6 inhibitor combination therapy exhibited significant anti-angiogenic and anti-proliferative effects in experimental human melanomas, monitored by F-18-FDG-PET/CT and DW-MRI

Immunohistochemical Characterisation of Cell-Type Specific Expression of CK1δ in Various Tissues of Young Adult BALB/c Mice

BACKGROUND: Casein kinase 1 delta (CK1delta) phosphorylates many key proteins playing important roles in such biological processes as cell growth, differentiation, apoptosis, circadian rhythm and vesicle transport. Furthermore, deregulation of CK1delta has been linked to neurodegenerative diseases and cancer. In this study, the cell specific distribution of CK1delta in various tissues and organs of young adult BALB/c mice was analysed by immunohistochemistry. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical staining of CK1delta was performed using three different antibodies against CK1delta. A high expression of CK1delta was found in a variety of tissues and organ systems and in several cell types of endodermal, mesodermal and ectodermal origin. CONCLUSIONS: These results give an overview of the cell-type specific expression of CK1delta in different organs under normal conditions. Thus, they provide evidence for possible cell-type specific functions of CK1delta, where CK1delta can interact with and modulate the activity of key regulator proteins by site directed phosphorylation. Furthermore, they provide the basis for future analyses of CK1delta in these tissues

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo

Validierung der Casein Kinase 1 delta in vitro und in vivo

Mitglieder der Casein Kinase 1 (CK1) Familie, insbesondere CK1delta, sind an vielen regulatorischen Prozessen in der Zelle beteiligt. Daher kann eine Deregulation von CK1 Isoformen zur Entstehung von malignen Zellen beitragen. In dieser Arbeit wurde zum einen das therapeutische Potential von CK1-spezifischen Inhibitoren untersucht, zum anderen wurden die Auswirkungen der Veränderungen in der Aktivität von CK1delta auf die durch das große Tumorantigen (T-Ag oder T) des Simian Virus 40 (SV40) induzierte Tumorgenese des duktalen Mammakarzinoms in einem neu entwickelten WAP("whey acidic protein")-mutCK1delta/WAP-T bi-transgenen Mausmodell charakterisiert. Eine Inhibition von CK1delta/epsilon kann sowohl in vitro das Wachstum von etablierten Pankreastumorzelllinien hemmen, als auch in vivo zu einer Reduktion des Tumorwachstums führen. Zudem ergaben quantitative Genexpressionsanalysen, dass eine Behandlung der Tiere mit dem CK1 spezifischen Inhibitor IC261 oder Gemcitabin zu Veränderungen in der Expression von pro- und anti-apoptotischen Genen führt. Zudem charakterisierten wir den Einfluss von CK1delta auf die Transformationskompetenz von SV40 T-Ag in vitro und im Tiermodell. Wir konnten zeigen, dass die durch T-Ag induzierte Tumorgenese des Mammakarzinoms durch eine Koexpression von mutCK1delta und T-Ag in WAP-mutCK1delta/WAP-T bi-transgenen Tieren beeinflusst werden kann. Bi-transgene Tiere wiesen eine signifikant längere Lebensdauer auf als WAP-T transgene Tiere. In ersten molekulare Analysen der Tumoren aus WAP-T Tieren und WAP-mutCK1delta/WAP-T Tieren wurde die Expression verschiedener Gene, mittels Real-Time PCR analysiert. Hierbei zeigte sich ein signifikanter Unterschied in der Expression von am Wnt-Signalweg und an der DNA-Reparatur beteiligten Genen in Tumoren aus WAP-T und WAP-mutCK1delta/WAP Tieren. Unser bi-transgenes Mausmodell stellt eine hervorragende Möglichkeit dar, Progressions- und Regressionsfaktoren, welche an der Tumorgenese des Mammakarzinoms beteiligt sind, zu identifizieren