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Dye design can influence the ability of fluorescently labeled imaging agents to generate tumor contrast and has become an area of significant interest in the field of fluorescence-guided surgery (FGS). Here, we show that the charge-balanced near-infrared fluorescent (NIRF) dye FNIR-Tag enhances the imaging properties of a fluorescently labeled somatostatin analogue.

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein–coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody–drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580–90. ©2016 AACR.

Supplementary Fig. S1- Coomassie gel of anti-LGR5 mAb and anti-LGR5 ADCs. Supplementary Fig. S2- Confocal image of HEK293T cells with stable over-expression of hLGR4. Supplementary Fig. S3- Anti-LGR5 ADC binding assay and cytotoxicity of free MMAE. Supplementary Fig. S4- Anti-LGR5 mAb crossreactivity with mouse LGR5 and images of intestinal organoids treated with anti-LGR5 ADC or free MMAE. Supplementary Fig. S5- Representative images of LoVo xenograft tumors in mice from each treatment cohort and graph of average body weight. Supplementary Fig. S6- H&E staining of intestinal tissue sections. Supplementary Fig. S7. Images of Lovo xenograft tumor recurrence and weight.

Purpose: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid. Metronomic dosing, the frequent administration of chemotherapeutics at substantially lower than maximum tolerated doses (MTD), has been shown to result in reduced normal tissue toxicity and to minimize “off-treatment” exposure resulting in an improved therapeutic ratio.Experimental Design: We tested the hypothesis that hyaluronic acid (HA) conjugates of paclitaxel (TXL; HA-TXL) would exert strong antitumor effects with metronomic (MET) dosing and induce antiangiogenic effects superior to those achieved with MTD administration or with free TXL. Female nude mice bearing SKOV3ip1 or HeyA8 ovarian cancer cells were treated intraperitoneally (i.p.) with MET HA-TXL regimens (or MTD administration) to determine therapeutic and biologic effects.Results: All MET HA-TXL–treated mice and the MTD group revealed significantly reduced tumor weights and nodules compared with controls (all P values < 0.05) in the chemotherapy-sensitive models. However, the MTD HA-TXL–treated mice showed significant weight loss compared with control mice, whereas body weights were not affected in the metronomic groups in HeyA8-MDR model, reflecting reduced toxicity. In the taxane-resistant HeyA8-MDR model, significant reduction in tumor weight and nodule counts was noted in the metronomic groups whereas the response of the MTD group did not achieve significance. While both MTD and metronomic regimens reduced proliferation (Ki-67) and increased apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), only metronomic treatment resulted in significant reductions in angiogenesis (CD31, microvessel density). Moreover, metronomic treatment resulted in substantial increases in thrombospondin-1 (Tsp-1), an inhibitor of angiogenesis.Conclusions: This study showed that MET HA-TXL regimens have substantial antitumor activity in ovarian carcinoma, likely via a predominant antiangiogenic mechanism. Clin Cancer Res; 18(15); 4114–21. ©2012 AACR.

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Purpose:Clinically available intraoperative imaging tools to assist surgeons in identifying occult lesions are limited and partially responsible for the high rate of disease recurrence in patients with neuroendocrine tumors (NET). Using the established clinical efficacy of radiolabeled somatostatin analogs as a model, we demonstrate the ability of a fluorescent somatostatin analog to selectively target tumors that overexpress somatostatin receptor subtype-2 (SSTR2) and demonstrate utility for fluorescence-guided surgery (FGS).Experimental Design:A multimodality chelator (MMC) was used as a “radioactive linker” to synthesize the fluorescently labeled somatostatin analog, 67/68Ga-MMC(IR800)-TOC. In vivo studies were performed to determine the pharmacokinetic profile, optimal imaging time point, and specificity for SSTR2-expressing tissues. Meso- and microscopic imaging of resected tissues and frozen sections were also performed to further assess specific binding, and binding to human NETs was examined using surgical biospecimens from patients with pancreatic NETs.Results:Direct labeling with 67Ga/68Ga provided quantitative biodistribution analysis that was in agreement with fluorescence data. Receptor-mediated uptake was observed in vivo and ex vivo at the macro-, meso-, and microscopic scales. Surgical biospecimens from patients with pancreatic NETs also displayed receptor-specific agent binding, allowing clear delineation of tumor boundaries that matched pathology findings.Conclusions:The radioactive utility of the MMC allowed us to validate the binding properties of a novel FGS agent that could have a broad impact on cancer outcomes by equipping surgeons with real-time intraoperative imaging capabilities.

PDF file, 366KB, Effects of MET and MTD HA-TXL treatment in the HeyA8-MDR model on biological endpoints.

PDF file, 104KB, Effects of MET and MTD HA-TXL therapy on tumor nodules in ovarian cancer models.

Metastasis is the leading cause of cancer-related deaths, and metastatic cancers remain largely incurable due to chemoresistance. Biomarkers of metastatic cells are lacking, and probes that could be used to detect and target metastases would be highly valuable. Here we hypothesize that metastatic cancer cells express cell-surface receptors that can be harnessed for identification of molecules homing to metastases. Screening a combinatorial library in a mouse mammary tumor model of spontaneous metastasis identified cyclic peptides with tropism for cancer cells disseminated to the lungs. Two lead peptides, CLRHSSKIC and CRAGVGRGC, bound murine and human cells derived from breast carcinoma and melanoma in culture and were selective for metastatic cells in vivo. In mice, peptide CRAGVGRGC radiolabeled with 67Ga for biodistribution analysis demonstrated selective probe homing to lung metastases. Moreover, systemic administration of 68Ga-labeled CRAGVGRGC enabled noninvasive imaging of lung metastases in mice by PET. A CRAGVGRGC-derived peptide induced apoptosis upon cell internalization in vitro and suppressed metastatic burden in vivo. Colocalization of CLRHSSKIC and CRAGVGRGC with N-cadherin+/E-cadherin− cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition. We conclude that CRAGVGRGC is useful as a probe to facilitate the development of imaging modalities and therapies targeting metastases.Significance:This study identifies new molecules that bind metastatic cells and demonstrates their application as noninvasive imaging probes and vehicles for cytotoxic therapy delivery in preclinical cancer models.

Supplementary figures and tables. "Clean" version.

PDF file, 67KB, Effects of HA-TXL MTD and HA-TXL MET on white blood count (WBC)and hemoglobin levels.

Pancreatic neuroendocrine tumors (PNETs) are a rare but increasingly more prevalent cancer with heterogeneous clinical and pathological presentation. Surgery is the preferred treatment for all hormone-expressing PNETs and any PNET greater than 2 cm, but difficulties arise when tumors are multifocal, metastatic, or small in size due to lack of effective surgical localization. Existing techniques such as intraoperative ultrasound provide poor contrast and resolution, resulting in low sensitivity for such tumors.Somatostatin receptor type 2 (SSTR2) is commonly overexpressed in PNETs and presents an avenue for targeted tumor localization. SSTR2 is often used for pre-operative imaging and therapeutic treatment, with recent studies demonstrating that somatostatin receptor imaging (SRI) can be applied in radioguided surgery to aid in removal of metastatic lymph nodes and achieving negative surgical margins. However not all PNETs express SSTR2, indicating labeled SRI could benefit from using a supplemental label-free technique such as multiphoton microscopy (MPM), which has proven useful in improving the accuracy of diagnosing more common exocrine pancreatic cancers.Our work tests the suitability of combined SRI and MPM for localizing PNETs by imaging and comparing samples of PNETs and normal pancreatic tissue. Specimens were labeled with a novel SSTR2-targeted contrast agent and imaged using fluorescence microscopy, and subsequently imaged using MPM to collect four autofluo-rescent channels and second harmonic generation. Our results show that a combination of both SRI and MPM provides enhanced contrast and sensitivity for localizing diseased tissue, suggesting that this approach could be a valuable clinical tool for surgical localization and treatment of PNETs.

Metastasis is the leading cause of cancer-related deaths, and metastatic cancers remain largely incurable due to chemoresistance. Biomarkers of metastatic cells are lacking, and probes that could be used to detect and target metastases would be highly valuable. Here we hypothesize that metastatic cancer cells express cell-surface receptors that can be harnessed for identification of molecules homing to metastases. Screening a combinatorial library in a mouse mammary tumor model of spontaneous metastasis identified cyclic peptides with tropism for cancer cells disseminated to the lungs. Two lead peptides, CLRHSSKIC and CRAGVGRGC, bound murine and human cells derived from breast carcinoma and melanoma in culture and were selective for metastatic cells in vivo. In mice, peptide CRAGVGRGC radiolabeled with 67Ga for biodistribution analysis demonstrated selective probe homing to lung metastases. Moreover, systemic administration of 68Ga-labeled CRAGVGRGC enabled noninvasive imaging of lung metastases in mice by PET. A CRAGVGRGC-derived peptide induced apoptosis upon cell internalization in vitro and suppressed metastatic burden in vivo. Colocalization of CLRHSSKIC and CRAGVGRGC with N-cadherin+/E-cadherin− cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition. We conclude that CRAGVGRGC is useful as a probe to facilitate the development of imaging modalities and therapies targeting metastases. Significance: This study identifies new molecules that bind metastatic cells and demonstrates their application as noninvasive imaging probes and vehicles for cytotoxic therapy delivery in preclinical cancer models.

ObjectiveEnhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described.DesignWe performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin.ResultsWe demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such asASCL2andFZD10,was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFβi, mTORi and SRCi) for EpiC groups.ConclusionOur data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

Molecular imaging is an emerging non-invasive method to qualitatively and quantitively visualize and characterize biological processes. Among the imaging modalities, PET/SPECT and near-infrared (NIR) imaging provide synergistic properties that result in deep tissue penetration and up to cell-level resolution. Dual-modal PET/SPECT-NIR agents are commonly combined with a targeting ligand (e.g., antibody or small molecule) to engage biomolecules overexpressed in cancer, thereby enabling selective multimodal visualization of primary and metastatic tumors. The use of such agents for (i) preoperative patient selection and surgical planning and (ii) intraoperative FGS could improve surgical workflow and patient outcomes. However, the development of targeted dual-modal agents is a chemical challenge and a topic of ongoing research. In this review, we define key design considerations of targeted dual-modal imaging from a topological perspective, list targeted dual-modal probes disclosed in the last decade, review recent progress in the field of NIR fluorescent probe development, and highlight future directions in this rapidly developing field.

Background. Although therapeutic advances have led to enhanced survival in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, detection of residual disease remains challenging. Here, we examine two approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, as potential candidates for the development of immunoconjugates for fluorescence-guided surgery (FGS). Methods. mAbs were conjugated to the near-infrared fluorescent (NIRF) dye, IRDye800, and for quantitative in vitro assessment, to the radiometal chelator, desferrioxamine, to enable dual labeling with 89Zr. In vitro binding was evaluated in HER2-overexpressing (BT474, SKBR3) and HER2-negative (MCF7) cell lines. BT474 and MCF7 xenografts were used for in vivo and ex vivo fluorescence imaging. Results. In vitro findings demonstrated HER2-mediated binding for both fluorescent immunoconjugates and were in agreement with radioligand assays using dual-labeled immunoconjugates. In vivo and ex vivo studies showed preferential accumulation of the fluorescently-labeled mAbs in tumors and similar tumor-to-background ratios. In vivo HER2 specificity was confirmed by immunohistochemical staining of resected tumors and normal tissues. Conclusions. We showed for the first time that fluorescent trastuzumab and pertuzumab immunoconjugates have similar NIRF imaging performance and demonstrated the possibility of performing HER2-targeted FGS with agents that possess distinct epitope specificity.

Significance: The use of cancer-targeted contrast agents in fluorescence-guided surgery (FGS) has the potential to improve intraoperative visualization of tumors and surgical margins. However, evaluation of their translational potential is challenging. Aim: We examined the utility of a somatostatin receptor subtype-2 (SSTR2)-targeted fluorescent agent in combination with a benchtop near-infrared fluorescence (NIRF) imaging system to visualize mouse xenografts under conditions that simulate the clinical FGS workflow for open surgical procedures. Approach: The dual-labeled somatostatin analog, Ga67-MMC(IR800)-TOC, was injected into mice (n = 24) implanted with SSTR2-expressing tumors and imaged with the customized OnLume NIRF imaging system (Madison, Wisconsin). In vivo and ex vivo imaging were performed under ambient light. The optimal dose (0.2, 0.5, and 2 nmol) and imaging time point (3, 24, 48, and 72 h) were determined using contrast-to-noise ratio (CNR) as the image quality parameter. Video captures of tumor resections were obtained to provide an FGS readout that is representative of clinical utility. Finally, a log-transformed linear regression model was fitted to assess congruence between fluorescence readouts and the underlying drug distribution. Results: The drug–device combination provided high in vivo and ex vivo contrast (CNRs > 3, except lung at 3 h) at all time points with the optimal dose of 2 nmol. The optimal imaging time point was 24-h post-injection, where CNRs > 6.5 were achieved in tissues of interest (i.e., pancreas, small intestine, stomach, and lung). Intraoperative FGS showed excellent utility for examination of the tumor cavity pre- and post-resection. The relationship between fluorescence readouts and gamma counts was linear and strongly correlated (n = 334, R2 = 0.71; r = 0.84; P

Hyaluronic acid (HA) with one reactive moiety grafted to the backbone is a commonly used matrix in tissue engineering. The addition of a second orthogonal moiety to the backbone allows for greater control in bioactive signal tethering and gelation. In this study, thiol and azide functional groups were grafted to the HA backbone at separate modification sites. NMR, FT-IR, colorimetric assay, and radio-TLC activity were used to confirm and quantify thiol and azide grafting to the HA backbone. Various ratios of di-functional HA (dif HA) and methacrylate HA (mHA) were used to encapsulate mouse embryonic stem cells in order to examine the neural differentiation of the cells. Greater neural maturation was observed in hydrogels containing a higher percentage of dif HA compared to mHA over a six day neural differentiation time course. This formulation was then tested in a contusion spinal cord injury model for biological effect and was found to reduce the ED1+ area in the spinal cord compared to control and allow for host axon extension into the matrix filled lesion area. These results indicate that dif HA is supportive of neural differentiation and can reduce inflammation without additional bioactive signal tethering. dif HA is a promising matrix base for the central nervous system, which should be further developed.

INTRODUCTION: The clinical need for improved intraoperative tumor visualization has led to the development of targeted contrast agents for fluorescence-guided surgery (FGS). A key characteristic of these agents is their high tumor specificity, which could enable detection of residual lesions that would likely be missed by visual inspection. Here, we examine the utility of a promising somatostatin receptor subtype-2 (SSTR2)-targeted fluorescent agent for detecting residual disease in mouse xenografts using FGS and post-operative histopathological validation. METHODS: Mice (n=2) implanted with SSTR2 overexpressing tumors were injected with 2 nmol of the dual-labeled somatostatin analog, (67)Ga-MMC(IR800)-TOC, and tumors were resected 48 h post-injection using traditional white light reflectance and palpation. Tumors underwent gamma counting and histopathology analysis. The wide-field FGS imaging platform (OnLume) was used to evaluate residual disease in situ under ambient light representative of an operating room. RESULTS: The tumor was resected with grossly negative margins using conventional inspection and palpation; however, additional in situ residual disease was found in the tumor cavity using FGS imaging. In situ fluorescent tumor contrast-to-noise ratios (CNRs) were 3.0 and 5.2. Agent accumulation was 7.72 and 8.20 %ID/g in tumors and 0.27 and 0.20 %ID/g in muscle. Fluorescence pixel values and gamma counts were highly correlated (r = 0.95, P < 0.048). H&E and IHC staining confirmed cancer positivity and SSTR2-overexpression, respectively. CONCLUSION: Our findings demonstrate that the use of clinically relevant fluorescence imaging instrumentation enhances the evaluation of promising FGS agents for in situ visualization of residual disease.

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss of TP53 and RB1 in both NEC tumors. To demonstrate an application of these novel NEC PDX models for SSTR2-targeted peptide imaging, the NEC913 and NEC1452 cells were bilaterally injected into mice. Near infrared-labelled octreotide was administered and the fluorescent signal was specifically observed for the NEC913 SSTR2 positive tumors. These 2 GEP NEC PDX models serve as a valuable resource for GEP NEN therapy testing.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is highly expressed in colorectal tumors and marks colon cancer stem cells that drive tumor growth and metastasis. Recently, we showed that LGR5 is a promising target for antibody–drug conjugate (ADC) therapy. However, it is important to identify LGR5-positive tumors that would respond to ADC treatment. Prior to drug conjugation, we evaluated two different anti-LGR5 monoclonal antibodies (mAbs), 8F2 and 9G5, using 89Zr-immunoPET to select the optimal mAb for ADC development and tumor imaging. Binding, specificity, and internalization were compared, and mAbs were prescreened as ADC candidates against colon cancer cells using secondary ADCs. Both mAbs demonstrated strong, specific binding in 293T-LGR5 cells but not 293T-vector cells. In DLD-1 colorectal cancer cells, which express high levels of LGR5, the mAbs rapidly internalized into lysosomes and promoted ADC-induced cytotoxicity, with 8F2 exhibiting slightly higher potency. No binding wa...

Background: Although advances in neoadjuvant and adjuvant treatments have led to enhanced survival in patients with HER2 positive breast cancer, intraoperative detection of residual disease and tumor margins (including HER2 positive DCIS) remain challenging. Real-time fluorescence imaging is being used during surgery to improve tumor visualization and identify margins. However, the lack of an approved intraoperative imaging agent that is specific for HER2 expressing cancers limits the utility of fluorescence-guided surgery (FGS) in this patient population. We examined the potential of using clinically approved anti-HER2 monoclonal antibodies (mAbs), trastuzumab and pertuzumab, for the development of fluorescent immunoconjugates dual-labeled with radioactivity to enable quantitative assessment. Methods: Trastuzumab, and pertuzumab were conjugated to the near-infrared (NIR) fluorophore, IR800CW, and radiometal chelator, deferoxamine (DFO), to permit dual labeling with 89Zr (positron emitting radionuclide). To confirm retention of HER2 specificity and functionality, flow cytometry analysis of the dual-labeled immunoconjugates was performed in HER2+ (BT474, SKBR3), HER2 moderate (MCF7), and HER2− (MDA231) cell lines. Using the same cell lines, radioactive uptake studies were employed to quantitatively measure cellular uptake study using 89Zr with and without blocking doses of unlabeled mAb. In vivo optical imaging of each immunoconjugate and IgG controls was performed in BT474 and MCF7 xenografts in BALB/c (nu/nu) mice 48 hours post injection using the BrukerXtreme system. Results: In vitro results confirmed retention of receptor mediated binding on both flow cytometry and radioactive analyses. As shown in Table 1, uptake of each immunoconjugate correlated with the degree of HER2 expression. Importantly, the addition of unlabeled mAb led to a 79-93% reduction in binding to cells with the highest HER2 expression (BT474 and SKBR3). In MDA231 cells binding of the immunoconjugates was similar to the IgG control and indicates low non-specific binding. In vivo and ex vivo analysis of HER2+ and HER2 moderate xenografts demonstrated prominent tumor uptake with minimal signal in non-clearance organs. As a result, tumor-to-muscle, tumor-to-fat pad, and tumor-to-lung ratios were >5, indicating excellent contrast in key regions where surgical excisions are performed clinically. Table 1: Uptake of 89Zr-mAb-IR800 in breast cancer cell linesCell Lines% Uptake of 89Zr-PertuzumabPertuzumab Block (Unlabeled)% Uptake of 89Zr-TrastuzumabTrastuzumab Block (Unlabeled)% Uptake of 89Zr-IgGBT47449.2±14.83.7±0.036.9±5.84.6±2.61.3±0SKBR337.9±1.77.9±0.942.8±3.52.9±1.73.5±0.4MCF721.7±2.63.2±0.44.0±0.12.5±0.11.3±0.7MDA2311.9±0.40.8±0.31.0±0.31.9±0.51.3±0.7 Conclusion: This study demonstrates the potential utility of HER2-targeted FGS to improve tumor visualization, increase the likelihood of obtaining clean surgical margins, and ultimately reduce morbidity in patients with HER2 breast cancer. Citation Format: Jo Simien, Julie Voss, A.M. Thompson, S.C. Ghosh, S. AghaAmiri, A. Azhdarinia, H.S. Tran-Cao. HER2/neu antibody-fluorophore conjugate for intraoperative detection of HER2-positive breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-02-01.

Introductory statement: To overcome enzyme-mediated resistance mechanisms associated with the DNA alkylating agent, temozolomide (TMZ), we seek to develop a peptide-drug conjugate (PDC) for selective drug delivery in cells that express the somatostatin receptor subtype-2 (SSTR2). Material and methods: The clinically used somatostatin analog, 68Ga-DOTA-TOC, was used as a model for development of the SSTR2-targeted PDC. Synthesis was performed by (i) replacing DOTA with a multimodality chelator (MMC), (ii) attaching a modified TMZ analog to MMC, and (iii) conjugating the payload moiety to TOC on solid-phase. The resulting product, MMC(TMZ)-TOC, was radiolabeled with 67Ga using cation exchange chromatography methods optimized for 68Ga. Retention of SSTR2 binding was examined in cell lines with different expression level of SSTR2 with and without blocking doses of octreotide. A membrane acid wash was performed to evaluate internalization efficiency. The cytotoxicity of MMC(TMZ)-TOC was tested in IMR-32 cells that endogenously express SSTR2, and potency was compared to free-TMZ. An alkaline comet assay was then performed to assess the DNA-damaging properties of the PDC in the presence and absence of SSTR2 blocking. Unpublished results: MMC(TMZ)-TOC was efficiently produced with chemical purity >90% as shown by high-performance liquid chromatography (HPLC). Radiochemical purity following 67Ga labeling was >95% and indicates the feasibility of using the MMC to directly label the drug conjugates. Cell-based experiments showed that specific binding of the 67Ga-labeled PDC was similar to 67Ga-DOTA-TOC and correlated with SSTR2 expression. In HCT116-SSTR2 cells that overexpress SSTR2, 14.8±4.8% of 67Ga-MMC(TMZ)-TOC and 17.0±4.2% of 67Ga-DOTA-TOC were taken up by cells. Blocking the excess octreotide reduced binding to near background levels, illustrating receptor-mediated uptake of the PDC. Acid-washing demonstrated internalization of 67Ga-MMC(TMZ)-TOC after receptor-binding, indicating retention of the agonist properties of TOC after payload conjugation. Results from the cell cytotoxicity study demonstrated that the PDC inhibited cell growth in a dose-dependent manner that was similar to free-TMZ, with the IC50 values of 81.6 and 75.6 µM for free-TMZ and MMC(TMZ)-TOC, respectively. The comet assay revealed that MMC(TMZ)-TOC was effective in causing DNA damage in cells that express SSTR2, as shown by reduced cytotoxic effects when co-incubated with octreotide. Conclusions: We showed that a clinically used SSTR2-targeted radiopharmaceutical can be converted into a PDC that retains receptor-binding and internalization properties. We also showed that the cytotoxic effects of TMZ are maintained by the PDC. Based on these results, in vivo evaluation of MMC(TMZ)-TOC is warranted to assess its potential as a targeted therapy for NETs. Citation Format: Solmaz AghaAmiri, Sukhen C. Ghosh, Lea Stitzlein, Servando Hernandez Vargas, Bruno Perlatti, Daniel M. Halperin, Ali Azhdarinia. A novel peptide-drug conjugate induces DNA damage in SSTR2-expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB120.

Intraoperative detection of tumors has had a profound impact on how cancer surgery is performed and addresses critical unmet needs in surgical oncology. Tumor deposits, margins, and residual cancer can be imaged through the use of fluorescent contrast agents during surgical procedures to complement visual and tactile guidance. The combination of fluorescent and nuclear contrast into a multimodality agent builds on these capabilities by adding quantitative, noninvasive nuclear imaging capabilities to intraoperative imaging. This review focuses on new strategies for the development and evaluation of targeted dual-labeled molecular imaging agents while highlighting the successful first-in-human application of this technique.

Multimodal imaging agents were first introduced a decade ago and consist of a targeting moiety that is dual-labeled with radioactive and fluorescent contrast. These compounds allow whole-body and intraoperative imaging to be performed through administration of a single agent and provide complementary diagnostic information that can be used to guide tumor resection. Since their initial evaluation, interest in dual-labeled agents has continued to grow and their design has subsequently evolved alongside the development of novel chelating agents, improved fluorophores, and highly selective coupling techniques for bioconjugate formation. In this review, will discuss how changes in the labeling components and schemes for multimodal agent development have impacted imaging performance and will focus on antibody- and peptide-based agents as models for dual labeling. We will also describe the growing role of modular dual labeling strategies as well as direct labeling methods using radiohalogens.

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1–4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing β-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.

A water-soluble octanuclear cluster, [Fe 8 ], was studied with regard to its properties as a potential contrast enhancing agent in magnetic resonance imaging (MRI) in magnetic fields of 1.3, 7.2 and 11.9 T and was shown to have transverse relaxivities r 2 = 4.01, 10.09 and 15.83 mM s −1 , respectively. A related hydrophobic [Fe 8 ] cluster conjugated with 5 kDa hyaluronic acid (HA) was characterized by 57 Fe-Mossbauer and MALDI-TOF mass spectroscopy, and was evaluated in aqueous solutions in vitro with regard to its contrast enhancing properties [r 2 = 3.65 mM s −1 (1.3 T), 26.20 mM s −1 (7.2 T) and 52.18 mM s −1 (11.9 T)], its in vitro cellular cytotoxicity towards A-549 cells and COS-7 cells and its in vivo enhancement of T 2 -weighted images (4.7 T) of a human breast cancer xenografted on a nude mouse. The physiologically compatible [Fe 8 ]-HA conjugate was i.v. injected to the tumor-bearing mouse, resulting in observable, heterogeneous signal change within the tumor, evident 15 min after injection and persisting for approximately 30 min. Both molecular [Fe 8 ] and its HA-conjugate show a strong magnetic field dependence on r 2 , rendering them promising platforms for the further development of T 2 MRI contrast agents in high and ultrahigh magnetic fields.

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog

Passive protection, the administration of antibodies to prevent infection, has garnered significant interest in recent years as a potential prophylactic countermeasure to decrease the prevalence of hospital-acquired infections. Pili, polymerized protein structures covalently anchored to the peptidoglycan wall of many Gram-positive pathogens, are ideal targets for antibody intervention, given their importance in establishing infection and their accessibility to antibody interactions. In this work, we demonstrated that a monoclonal antibody to the major component of Enterococcus faecalis pili, EbpC, labels polymerized pilus structures, diminishes biofilm formation, and significantly prevents the establishment of a rat endocarditis infection. The effectiveness of this anti-EbpC monoclonal provides strong evidence in support of its potential as a preventative. In addition, after radiolabeling, this monoclonal identified the site of enterococcal infection, providing a rare example of molecularly specific imaging of an established bacterial infection and demonstrating the versatility of this agent for use in future diagnostic and therapeutic applications.

Wide-field surgical excision reduces the chance of residual disease, but can also lead to disfigurement and devastating morbidities when resection is close to critical structures. We hypothesize that near-infrared fluorescence (NIRF) imaging can enable accurate detection of tumor margins for image-guided resection. An orthotopic model of human prostate cancer (PCa) was used to assess primary tumor margins using a NIRF-labeled antibody against epithelial cell adhesion molecule (EpCAM). PCa cells stably expressing far red fluorescent gene reporter, iRFP, enabled colocalization with NIRF signals for direct assessment of tumor margins. Using receiver operating characteristic analysis, far red fluorescence was validated against standard pathology of primary and metastatic lesions with >96 % accuracy. Primary tumor margins were more accurately detected by quantitative NIRF imaging using the EpCAM-targeting antibody as compared to a NIRF-labeled isotype control antibody. NIRF molecular imaging may enable real-time and accurate assessment of tumor margins.

Dual-labeled compounds containing nuclear and near-infrared fluorescence contrast have the potential to molecularly guide surgical resection of cancer by extending whole-body diagnostic imaging findings into the surgical suite. To simplify the dual labeling process for antibody-based agents, we designed a multimodality chelation (MMC) scaffold which combined a radiometal chelating agent and fluorescent dye into a single moiety. Three dye-derivatized MMC compounds were synthesized and radiolabeled. The IRDye 800CW conjugate, 4, had favorable optical properties and showed rapid clearance in vivo. Using 4, an epithelial cell adhesion molecule (EpCAM) targeting MMC-immunoconjugate was prepared and dual-labeled with (64)Cu. In vitro binding activity was confirmed after MMC conjugation. Multimodal imaging studies showed higher tumor accumulation of (64)Cu-7 compared to nontargeted (64)Cu-4 in a prostate cancer model. Further evaluation in different EpCAM-expressing cell lines is warranted as well as application of the MMC dual labeling approach with other monoclonal antibodies.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

Gastrointestinal cancer is one of the leading causes of cancer-related mortality in men and women worldwide. The adult stem cell marker LGR5 (leucine-rich repeat-containing, G protein–coupled receptor 5) is highly expressed in a significant fraction of gastrointestinal tumors of the colon, liver, pancreas, and stomach, relative to normal tissues. LGR5 is located on the cell surface and undergoes rapid, constitutive internalization independent of ligand. Furthermore, LGR5-high cancer cells have been shown to exhibit the properties of tumor-initiating cells or cancer stem cells (CSC). On the basis of these attributes, we generated two LGR5-targeting antibody–drug conjugates (ADC) by tethering the tubulin-inhibiting cytotoxic drug monomethyl auristatin E to a highly specific anti-LGR5 mAb via a protease cleavable or noncleavable chemical linker and compared them in receptor binding, cell internalization, and cytotoxic efficacy in cancer cells. Here, we show that both ADCs bind LGR5 with high specificity and equivalent nanomolar affinity and rapidly internalize to the lysosomes of LGR5-expressing gastrointestinal cancer cells. The anti-LGR5 ADCs effectively induced cytotoxicity in LGR5-high gastrointestinal cancer cells, but not in LGR5-negative or -knockdown cancer cell lines. Overall, we demonstrate that the cleavable ADC exhibited higher potency in vitro and was able to eradicate tumors and prevent recurrence in a xenograft model of colon cancer. These findings provide preclinical evidence for the potential of LGR5-targeting ADCs as effective new therapeutics for the treatment and eradication of gastrointestinal tumors and CSCs with high LGR5 expression. Mol Cancer Ther; 15(7); 1580–90. ©2016 AACR.

Purpose: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid. Metronomic dosing, the frequent administration of chemotherapeutics at substantially lower than maximum tolerated doses (MTD), has been shown to result in reduced normal tissue toxicity and to minimize “off-treatment” exposure resulting in an improved therapeutic ratio. Experimental Design: We tested the hypothesis that hyaluronic acid (HA) conjugates of paclitaxel (TXL; HA-TXL) would exert strong antitumor effects with metronomic (MET) dosing and induce antiangiogenic effects superior to those achieved with MTD administration or with free TXL. Female nude mice bearing SKOV3ip1 or HeyA8 ovarian cancer cells were treated intraperitoneally (i.p.) with MET HA-TXL regimens (or MTD administration) to determine therapeutic and biologic effects. Results: All MET HA-TXL–treated mice and the MTD group revealed significantly reduced tumor weights and nodules compared with controls (all P values < 0.05) in the chemotherapy-sensitive models. However, the MTD HA-TXL–treated mice showed significant weight loss compared with control mice, whereas body weights were not affected in the metronomic groups in HeyA8-MDR model, reflecting reduced toxicity. In the taxane-resistant HeyA8-MDR model, significant reduction in tumor weight and nodule counts was noted in the metronomic groups whereas the response of the MTD group did not achieve significance. While both MTD and metronomic regimens reduced proliferation (Ki-67) and increased apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), only metronomic treatment resulted in significant reductions in angiogenesis (CD31, microvessel density). Moreover, metronomic treatment resulted in substantial increases in thrombospondin-1 (Tsp-1), an inhibitor of angiogenesis. Conclusions: This study showed that MET HA-TXL regimens have substantial antitumor activity in ovarian carcinoma, likely via a predominant antiangiogenic mechanism. Clin Cancer Res; 18(15); 4114–21. ©2012 AACR.

Advances in cancer therapeutics, namely more effective and less toxic treatments, will occur with targeting strategies that enhance the tumor biodistribution and thwart normal tissue exposure of the drug. This review focuses on cancer drug targeting approaches that exploit the expression of the cell-surface proteoglycan family, CD44, on the tumor cell surface followed by some form of ligand binding and induced CD44 internalization and intracellular drug release: in effect using this as a 'Trojan Horse' to more selectively access tumor cells.This review defines the origins of evidence for a linkage between CD44 expression and malignancy, and invokes contemporary views of the importance of putative CD44(+) cancer stem cells in disease resistance. Although the primary emphasis is on the most advanced and developed paths, those that have either made it to the clinic or are well-poised to get there, a wide scope of additional approaches at various preclinical stages is also briefly reviewed.The future should see development of drug targeting approaches that exploit CD44 expression on CSCs/TICs, including applications to cytotoxic agents currently in the clinic.

BACKGROUND: Although inflammatory breast cancer (IBC) is recognized as the most lethal variant of locally advanced breast cancer, few molecular signatures of IBC have been identified that can be used as targets to develop therapeutics that effectively inhibit the aggressive phenotype displayed by IBC tumors. METHODS: Real-time polymerase chain reaction analysis, Western blot analysis, modified Boyden chamber invasion assays, vasculogenic mimicry (VM) assays, and gelatin zymography were used in the current studies. Agonists and antagonists of the prostanoid receptors EP3 and EP4 and of EP4 short-hairpin RNA (shRNA) knockdown approaches were used as tools to assess the role of prostanoid receptors EP3 and EP4 in the regulation of specific biologic activities of IBC cells. RESULTS: The current studies revealed that the IBC breast cancer cell lines SUM149 and SUM190 express high levels of cyclooxygenase-2 messenger RNA and protein, produce abundant levels of prostaglandin E2, and produce both EP3 and EP4 receptor proteins. Studies using the EP4 antagonist GW627368X and shRNA molecular knockdown approaches revealed a role for EP4 in regulating invasion of IBC cells. EP3, but not EP4, regulated the ability of SUM149 cells to undergo VM, which is the ability to form capillary-like structures, a characteristic exhibited by very aggressive tumor types. Inhibition of VM by sulprostone was associated with an inhibition of matrix metalloprotease-2 (MMP-2) enzyme activity. CONCLUSIONS: The prostanoid receptors EP3 and EP4 differentially regulate activities exhibited by IBC cells that have been associated with the aggressive phenotype of this lethal variant of breast cancer. Whereas EP4 regulates invasion, EP3 regulates VM and the associated increased MMP-2 enzyme activity. Cancer 2010;116(11 suppl):2806–14. © 2010 American Cancer Society.

Multidrug-resistance-associated protein (MRP) and BCL-2 contribute to drug resistance expressed in SCLC. To establish whether MRP-mediated drug resistance affects sphingolipid (SL)-induced apoptosis in SCLC, we first examined the human SCLC cell line, UMCC-1, and its MRP over-expressing, drug-resistant subline, UMCC-1/VP. Despite significantly decreased sensitivity to doxorubicin (Dox) and to the etoposide, VP-16, the drug-selected line was essentially equally as sensitive to treatment with exogenous ceramide (Cer), sphingosine (Sp) or dimethyl-sphingosine (DMSP) as the parental line. Next, we observed that high BCL-2-expressing human H69 SCLC cells, that were approximately 160-fold more sensitive to Dox than their combined BCL-2 and MRP-over-expressing (H69AR) counterparts, were only approximately 5-fold more resistant to DMSP. Time-lapse fluorescence microscopy of either UMCC cell line treated with DMSP-Coumarin revealed comparable extents and kinetics of SL uptake, further ruling out MRP-mediated effects on drug uptake. DMSP potentiated the cytotoxic activity of VP-16 and Taxol, but not Dox, in drug-resistant UMCC-1/VP cells. However, this sensitization did not appear to involve DMSP-mediated effects on the function of MRP in drug export; nor did DMSP strongly shift the balance of pro-apoptotic Sps and anti-apoptotic Sp-1-Ps in these cells. We conclude that SL-induced apoptosis markedly overcomes or bypasses MRP-mediated drug resistance relevant to SCLC and may suggest a novel therapeutic approach to chemotherapy for these tumors.

Poly- l -glutamic acid (PGA) has previously been demonstrated to be an effective backbone for creating a hydrophilic prodrug of the established anti-tumor agent, paclitaxel, the active agent in Taxol; this approach has obviated the need for the toxic Cremophor excipient, used to enhance the solubility of paclitaxel in the clinical formulation. In order to form hydrophilic prodrugs of the hydrophobic pro-apoptotic sphingolipid, N,N -dimethylsphingosine (DMSP), PGA was condensed with DMSP, previously modified with coumarin to allow spectroscopic detection during conjugate synthesis, to yield PGA–DMSP. Conjugates with different loadings of DMSP were prepared and evaluated for in vitro cytotoxicity against two human breast adenocarcinoma cell lines. Time- and loading-dependent expression of cytotoxicity was observed, such that endpoints essentially equivalent to those observed with free-DMSP were achieved, but in a more protracted manner, consistent with prodrug behavior. PGA–DMSP was initially evaluated for toxicity in female nude mice, and administration of high net levels of DMSP, exceeding those achievable with free-DMSP, was well-tolerated. We propose that PGA–DMSP conjugates merit evaluation for anti-tumor efficacy in pre-clinical tumor models.

Numerous human tumor types, including ovarian cancer, display a significant expression of the CD44 family of cell surface proteoglycans. To develop tumortargeted drugs, we have initially evaluated whether the CD44 ligand hyaluronic acid (HA) could serve as a backbone for paclitaxel (TXL) prodrugs. HA-TXL was prepared by modification of previous techniques. The in vitro cytotoxicity of HA-TXL against the CD44(+) human ovarian carcinoma cell lines SKOV-3ip and NMP-1 could be significantly blocked by preincubation with a molar excess of free HA. Female nude mice bearing intraperitoneal implants of NMP-1 cells were treated intraperitoneally with a single sub-maximum tolerated dose dose of HA-TXL or with multiple-dose regimens of paclitaxel (Taxol; Mead Johnson, Princeton, NJ) to determine the effects of these regimens on host survival and intraperitoneal tumor burden, with the latter being assessed by magnetic resonance imaging. NMP-1 xenograffs were highly resistant to Taxol regimens, as host survival was only nominally improved compared to controls (T/C ∼ 120), whereas singledose HA-TXL treatment significantly improved survival in this model (T/C ∼ 140; P = .004). In both NMP-1 and SKOV-3ip models, MR images of abdomens of HA-TXL-treated mice obtained shortly before controls required humane sacrifice revealed markedly reduced tumor burdens compared to control mice. This study is among the first to demonstrate that HA-based prodrugs administered locoregionally have antitumor activity in vivo.

Coumarin derivatives of N,N-dimethylsphingosine (DMSP) were prepared and chemically characterized. They were apparently biologically equivalent to DMSP in terms of tumor cell cytotoxicity and were used to establish the rapid mitochondrial localization of this sphingolipid in tumor cells, followed closely by its marked reduction of mitochondrial membrane potential.

The feasibility of applying magnetic resonance imaging (MRI) for conducting prospective studies of intraperitoneal (i.p.) tumor treatment response to chemotherapy and resultant effects on survival in human ovarian carcinoma/nude mouse orthotopic xenograft models was evaluated. Female nude mice were implanted i.p. with either NMP-1 or SKOV-3ip. human ovarian carcinoma cells on day 0. Initial T2-weighted magnetic resonance (MR) images of the abdomens of NMP-1–implanted mice were obtained on day 7 to confirm the presence of nascent tumors; similar confirmations were made on day 14 with mice bearing SKOV-3ip. xenografts. On the initial imaging days, a multiple-dose regimen of cisplatin (CDDP; qd7 ×3) was commenced, using 4 or 6 mg/kg treatments with the NMP-1 model and using 6 mg/kg treatments with the SKOV-3ip. model. Mice were reimaged multiple times, 2 days following each CDDP injection and at later times as well, depending on host survival. The images for each mouse from the last imaging day (day 30 for NMP-1, day 44 for SKOV-3ip.) were used in a blinded fashion to attempt to visually distinguish control from treated mice and to determine whether MRI could predict a survival benefit. For SKOV-3ip. mice, ten out of ten mice were correctly segregated into the control or the CDDP treatment group based solely on these blinded, nonquantified MR results. In this model, the 6 mg/kg multiple-dose regimen achieved a modest response, improving life span by ∼24%. However, for the NMP-1 mice, only six out of nine evaluable mice were correctly segregated into the control or one of the treatment groups by similar MRI criteria, a virtually random distribution; further, neither CDDP treatment regimen achieved a significant improvement in survival in this model. In another study, NMP-1–implanted mice were treated on day 7 after tumor implantation with a single injection of a hyaluronic acid–paclitaxel copolymer. Control and treated mice were MR imaged on day 28, which revealed marked reductions in tumor burden in treated mice, correlating well with a subsequently observed improved survival of ∼40%. Our results suggest that MRI can be used to serially and noninvasively monitor treatment response and predict ongoing treatment effects on survival.

Introduction Bifunctional chelators have been shown to impact the biodistribution of monoclonal antibody (mAb)-based imaging agents. Recently, radiolabeled 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-peptide complexes have demonstrated improved in vivo stability and performance compared to their 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) counterparts. Here, we investigated if similar utility could be achieved with mAbs and compared 64 Cu-labeled DOTA and NODAGA-immunoconjugates for the detection of epithelial cell adhesion molecule (EpCAM) in a prostate cancer model. Methods DOTA and NODAGA-immunoconjugates of an EpCAM targeting mAb (mAb7) were synthesized and radiolabeled with 64 Cu (DOTA: 40°C for 1hr; NODAGA: 25°C for 1hr). The average number of chelators per mAb was quantified by isotopic dilution, and the biological activity of the immunoconjugates was evaluated by flow cytometry and ELISA. Radioligand assays were performed to compare cellular uptake and determine the dissociation constant ( K d ) and maximum number of binding sites ( B max ) for the immunoconjugates using DsRed-transfected PC3-cells. A PC3-DsRed xenograft tumor model was established in nude mice and used to perform biodistribution studies to compare organ uptake and pharmacokinetics. Results 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7 were prepared with chelator/protein ratios of 2–3 and obtained in comparable radiochemical yields ranging from 59 to 71%. Similar immunoreactivity was observed with both agents, and mock labeling studies indicated that incubation at room temperature or 40°C did not affect potency. 64 Cu-NODAGA-mAb7 demonstrated higher in vitro cellular uptake while 64 Cu-DOTA-mAb7 had higher K d and B max values. From the biodistribution data, we found similar tumor uptake (13.44±1.21%ID/g and 13.24±4.86%ID/g for 64 Cu-DOTA-mAb7 and 64 Cu-NODAGA-mAb7, respectively) for both agents at 24hr, although normal prostate tissue was significantly lower for 64 Cu-NODAGA-mAb7. 64 Cu-NODAGA-mAb7 also had less accumulation in the liver, suggesting excellent retention of the chelation complex in vivo . This was further confirmed by the higher blood activity of 64 Cu-NODAGA-mAb7, which corresponds to increased bioavailability afforded by the enhanced in vivo stability of the agent. Although tumor/muscle ratios were comparable, tumor/prostate ratios were >2-fold and 1.5-fold higher for 64 Cu-NODAGA-mAb7 at 24 and 48hr, respectively, and suggest better ability to discriminate tumor tissue with 64 Cu-NODAGA-mAb7 in our prostate cancer model. Conclusions To the best of our knowledge, this study represents the first comparison of 64 Cu-labeled DOTA and NODAGA immunoconjugates in vivo . Our results show favorable in vivo performance for 64 Cu-NODAGA-mAb7 which builds upon previous data on our hybrid mAb7 imaging agent by increasing the detection sensitivity for metastatic prostate tumors, as well as for other types of cancer that express EpCAM.

The presence of brown adipose tissue responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Owing to limitations of current methods for brown adipose tissue detection, analysing the abundance and localization of brown adipose tissue in the body has remained challenging. Here we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of brown adipose tissue, but not of intraperitoneal white adipose tissue. We show that in addition to brown adipose tissue, this peptide probe also recognizes the vasculature of brown adipose tissue-like depots of subcutaneous white adipose tissue. Our results indicate that the CPATAERPC peptide localizes to brown adipose tissue even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify brown adipose tissue depots in mice by whole-body near-infrared fluorescence imaging.

Nuclear and near-infrared fluorescence (NIRF) imaging provide non-invasive monitoring of disease. Merging the respective contrast agents through dual labeling could facilitate NIRF validation using proven nuclear imaging methodology while expanding the utility of diagnostic radiotracers.

Summary Chemotherapeutic regimens incorporating taxanes significantly improve outcomes for patients with squamous cell carcinomas of the head and neck (SCCHN). However, treatment with taxanes is limited by toxicities, including bone marrow suppression and peripheral neuropathies. We proposed that conjugating taxanes to targeting carrier molecules would increase antitumor efficacy and decrease toxicity. The cell surface proteoglycan, CD44, is expressed on most SCCHNs, and we hypothesized that it is an attractive candidate for targeted therapy via its natural ligand, hyaluronic acid (HA). We determined whether HA–paclitaxel conjugates were able to decrease tumor growth and improve survival in orthotopic nude mouse human SCCHN xenograft models. HA–paclitaxel concentration-dependent growth inhibition of human SCCHN cell lines OSC-19 and HN5 in vitro, very similarly to free paclitaxel treatment. Tumor cell uptake of FITC-labeled HA–paclitaxel was significantly blocked with free HA, indicating the dependence of uptake on CD44. HA–paclitaxel administered intravenously once per week for three weeks at 120 mg/kg paclitaxel equivalents, far above the paclitaxel maximum tolerated dose, exerted superior tumor growth control to that of paclitaxel in both orthotopic OSC-19-luciferase and HN5 xenograft models in vivo. Mouse survival following HA–paclitaxel administration was prolonged compared with that of controls in mice implanted with either of these xenografts. Mice treated with HA–paclitaxel displayed increased TUNEL(+) cells in tumor tissue, as well as markedly reduced microvessel density compared to those treated with free paclitaxel. No acute histopathological changes were observed in mice treated with HA–paclitaxel. Thus, we conclude that HA–paclitaxel effectively inhibits tumor growth in human SCCHN xenografts via an HA-mediated mechanism and this conjugate should be considered for further preclinical development for this disease.

BACKGROUND Elevated BCL-2 is one mechanism of therapeutic resistance in prostate cancer (PC), and new approaches are needed to overcome such resistance. METHODS We evaluated the effects of BCL-2 over-expression in human prostatic adenocarcinoma cells on their susceptibility to sphingolipids (SLs) and to the sphingosine kinase (SpK) inhibitor, SKI II. RESULTS In survival assays, no significant differences were observed in the responses to sphingosine or ceramide among parental PC-3 cells lacking detectable BCL-2 and BCL-2 over-expressing PC-3 transfectants; similarly, the responses to dimethyl-sphingosine (DMSP) of parental LNCaP cells and a BCL-2 over-expressing LNCaP transfectant were equivalent. SKI II induced protracted, BCL-2-independent survival loss in both PC-3 and LNCaP parental/transfectant pairs; in contrast, DMSP induced rapid cell shrinkage, caspase activation and caspase-dependent DNA fragmentation. DMSP-induced DNA fragmentation and loss of mitochondrial membrane potential were equivalent in BCL-2 transfectants and parental PC-3 cells and were not associated with BCL-2 downregulation. DMSP-mediated cytotoxicity was not associated with the enhanced production of reactive oxygen intermediates. SL analyses of parental and transfectant PC-3 cells did not reveal increased levels of sphingosine-1-phosphate in the BCL-2 transfectants; further, there only a modest early shift, corresponding to apoptotic onset, in pro- versus anti-apoptotic SLs in response to DMSP treatment. CONCLUSIONS Thus, in contrast to the inhibitory effects of BCL-2 on apoptosis induced by various agents in tumor cells, SKI II and selected pro-apoptotic SLs appear atypical in their independence from such inhibition, and may have merits as new candidates for treatment of AI PC. Prostate 67: 1699–1717, 2007. © 2007 Wiley-Liss, Inc.

Paclitaxel (Taxol) has demonstrated clinical activity in non–small-cell lung cancer (NSCLC), but its use has not led to marked improvements in survival. This ineffectiveness can in part be attributed to inadequate delivery of effective drug levels to the lung via systemic administration and to drug resistance mechanisms. Locoregional drug administration and the use of drug copolymers are possible approaches to address these issues. In this study, we evaluated the activity of a poly(l-glutamic acid)-paclitaxel (PGA-TXL) formulation administered by intratracheal injection to mice bearing orthotopic human NSCLC tumors (H460, H358). H460 cells were found to be sensitive to paclitaxel and PGA-TXL in vitro, in a time- and concentration-dependent manner. In preliminary acute toxicity studies, PGA-TXL administered by intratracheal injection was found to be much less toxic than paclitaxel, as anticipated. Mice into which H460 cells had been implanted by intratracheal injection were given single-dose intratracheal treatments with paclitaxel (1.2 or 2.4 mg/kg) or with PGA-TXL (15 mg/kg, paclitaxel equivalents) 1 week later. When the mice were sacrificed at up to 65 days after tumor implantation, they were evaluated grossly for tumor at bronchial, neck, and lung sites. Control mice had tumors in 60% of all three sites, and all of the control mice had tumors in at least one site. The low- and high-dose Taxol groups had fewer incidences at these three sites (27–33%) and 60–80% of these mice had tumors in at least one site. The PGA-TXL mice displayed a low (13%) incidence at these sites, and only 40% had detectable tumors. In a subsequent survival study with the intratracheal H358 model, control mice had a mean life span of 95 days, whereas both the intratracheal Taxol (2.5 mg/kg, every 7th day for three doses) and the intratracheal PGA-TXL (20 mg/kg, paclitaxel equivalents, every 7th day for three doses) groups had improved survival (mean life spans: 133.5 and 136.5 days, respectively). In pilot studies intended to compare the feasibility of the development of paclitaxel aerosols suitable for clinical application, based either on Cremophor solutions or on PGA backbones, only the latter gave acceptable particle size distributions and flow rates. These results encourage the development and application of Cremophor-free copolymer formulations of paclitaxel for locoregional treatment (e.g., as aerosol) of endobronchial malignant diseases.

Specifically designed series of molecules (landers), comprising a central rigid polyaromatic core and several spacers that decouple the board from the metallic surface, have been synthesized. UHV-STM studies, on one hand, have shown important distortions of the molecule by interaction with the substrate. On the other hand, surface restructuring results from the presence of the molecules.