Multifunctional nanobeacon for imaging Thomsen‐Friedenreich antigen‐associated colorectal cancer

This research aimed to validate the specificity of the newly developed nanobeacon for imaging the Thomsen‐Friedenreich (TF) antigen, a potential biomarker of colorectal cancer. The imaging agent is comprised of a submicron‐sized polystyrene nanosphere encapsulated with a Coumarin 6 dye. The surface of the nanosphere was modified with peanut agglutinin (PNA) and poly(N‐vinylacetamide (PNVA) moieties. The former binds to Gal‐β(1‐3)GalNAc with high affinity while the latter enhances the specificity of PNA for the carbohydrates. The specificity of the nanobeacon was evaluated in human colorectal cancer cells and specimens, and the data were compared with immunohistochemical staining and flow cytometric analysis. Additionally, distribution of the nanobeacon in vivo was assessed using an “intestinal loop” mouse model. Quantitative analysis of the data indicated that approximately 2 μg of PNA were detected for each milligram of the nanobeacon. The nanobeacon specifically reported colorectal tumors by recognizing the tumor‐specific antigen through the surface‐immobilized PNA. Removal of TF from human colorectal cancer cells and tissues resulted in a loss of fluorescence signal, which suggests the specificity of the probe. Most importantly, the probe was not absorbed systematically in the large intestine upon topical application. As a result, no registered toxicity was associated with the probe. These data demonstrate the potential use of this novel nanobeacon for imaging the TF antigen as a biomarker for the early detection and prediction of the progression of colorectal cancer at the molecular level.     

Epidermal growth factor receptor‐targeted molecular imaging of colorectal tumors: Detection and treatment evaluation of tumors in animal models

To overcome the problem of overlooking colorectal tumors, a new and highly sensitive modality of colonoscopy is needed. Moreover, it is also important to establish a new modality to evaluate viable tumor volume in primary lesions of colorectal cancer (CRC) during chemotherapy. Therefore, we carried out molecular imaging of colorectal tumors targeting epidermal growth factor receptor (EGFR), which is highly expressed on tumor cells, for evaluating chemotherapeutic efficacy and for endoscopic detection of colorectal adenomas. We first attempted to image five CRC cell lines with various levels of EGFR expression using an Alexa Fluor‐labeled anti‐EGFR monoclonal antibody (AF‐EGFR‐Ab). A strong fluorescence signal was observed in the cells depending on the level of EGFR expression. When nude mice xenografted with LIM1215 CRC cells, which highly express EGFR, were i.v. injected with AF‐EGFR‐Ab, a strong fluorescence signal appeared in the tumor with a high signal to noise ratio, peaking at 48 hours after injection and then gradually decreasing, as shown using an IVIS Spectrum system. When the xenografted mice were treated with 5‐fluorouracil, fluorescence intensity in the tumor decreased in proportion to the viable tumor cell volume. Moreover, when the colorectum of azoxymethane‐treated rats was observed using a thin fluorescent endoscope with AF‐EGFR‐Ab, all 10 small colorectal adenomas (≤3 mm) were detected with a clear fluorescence signal. These preliminary results of animal experiments suggest that EGFR‐targeted fluorescent molecular imaging may be useful for quantitatively evaluating cell viability in CRC during chemotherapy, and also for detecting small adenomas using a fluorescent endoscope.     

In vivo subcellular imaging of tumors in mouse models using a fluorophore‐conjugated anti‐carcinoembryonic antigen antibody in two‐photon excitation microscopy

Recently, there has been growing interest in applying fluorescence imaging techniques to the study of various disease processes and complex biological phenomena in vivo. To apply these methods to clinical settings, several groups have developed protocols for fluorescence imaging using antibodies against tumor markers conjugated to fluorescent substances. Although these probes have been useful in macroscopic imaging, the specificity and sensitivity of these methods must be improved to enable them to detect micro‐lesions in the early phases of cancer, resulting in better treatment outcomes. To establish a sensitive and highly specific imaging method, we used a fluorophore‐conjugated anti‐carcinoembryonic antigen (CEA) antibody to perform macroscopic and microscopic in vivo imaging of inoculated cancer cells expressing GFP with or without CEA. Macroscopic imaging by fluorescence zoom microscopy revealed that bio‐conjugation of Alexa Fluor 594 to the anti‐CEA antibody allowed visualization of tumor mass consisting of CEA‐expressing human cancer cells, but the background levels were unacceptably high. In contrast, microscopic imaging using a two‐photon excitation microscope and the same fluorescent antibody resulted in subcellular‐resolution imaging that was more specific and sensitive than conventional imaging using a fluorescence zoom microscope. These results suggest that two‐photon excitation microscopy in conjunction with fluorophore‐conjugated antibodies could be widely adapted to detection of cancer‐specific cell‐surface molecules, both in cancer research and in clinical applications.     

Predictive imaging of chemotherapeutic response in a transgenic mouse model of pancreatic cancer

The underglycosylated mucin 1 tumor antigen (uMUC1) is a biomarker that forecasts the progression of adenocarcinomas. In this study, we evaluated the utility of a dual‐modality molecular imaging approach based on targeting uMUC1 for monitoring chemotherapeutic response in a transgenic murine model of pancreatic cancer (KCM triple transgenic mice). An uMUC1‐specific contrast agent (MN‐EPPT) was synthesized for use with magnetic resonance imaging (MRI) and fluorescence optical imaging. It consisted of dextran‐coated iron oxide nanoparticles conjugated to the near infrared fluorescent dye Cy5.5 and to a uMUC1‐specific peptide (EPPT). KCM triple transgenic mice were given gemcitabine as chemotherapy while control animals received saline injections following the same schedule. Changes in uMUC1 levels following chemotherapy were monitored using T2‐weighted MRI and optical imaging before and 24 hr after injection of the MN‐EPPT. uMUC1 expression in tumors from both groups was evaluated by histology and qRT‐PCR. We observed that the average delta‐T2 in the gemcitabine‐treated group was significantly reduced compared to the control group indicating lower accumulation of MN‐EPPT, and correspondingly, a lower level of uMUC1 expression. In vivo optical imaging confirmed the MRI findings. Fluorescence microscopy of pancreatic tumor sections showed a lower level of uMUC1 expression in the gemcitabine‐treated group compared to the control, which was confirmed by qRT‐PCR. Our data proved that changes in uMUC1 expression after gemcitabine chemotherapy could be evaluated using MN‐EPPT‐enhanced in vivo MR and optical imaging. These results suggest that the uMUC1‐targeted imaging approach could provide a useful tool for the predictive assessment of therapeutic response.     

Clostridium perfringens enterotoxin C‐terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy‐resistant ovarian cancer

Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin‐3‐ and ‐4‐overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy‐naïve and chemotherapy‐resistant human ovarian cancer xenografts or patient‐derived xenografts (PDXs) were treated with the carboxyl‐terminal binding domain of the Clostridium perfringens enterotoxin (c‐CPE) conjugated to FITC (FITC‐c‐CPE) or the near‐infrared (NIR) fluorescent tag IRDye CW800 (CW800‐c‐CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC‐c‐CPE and the CW800‐c‐CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800‐c‐CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor‐to‐background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c‐CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c‐CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real‐time detection of micrometastatic ovarian disease overexpressing the Claudin‐3 and ‐4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.     

Detection of colorectal neoplasia: Combination of eight blood‐based,cancer‐associated protein biomarkers

Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed eight cancer‐associated protein biomarkers in plasma from subjects undergoing first time ever colonoscopy due to symptoms attributable to colorectal neoplasia. Plasma AFP, CA19‐9, CEA, hs‐CRP, CyFra21‐1, Ferritin, Galectin‐3 and TIMP‐1 were determined in EDTA‐plasma using the Abbott ARCHITECT® automated immunoassay platform. Primary endpoints were detection of (i) CRC and high‐risk adenoma and (ii) CRC. Logistic regression was performed. Final reduced models were constructed selecting the four biomarkers with the highest likelihood scores. Subjects (N = 4,698) were consecutively included during 2010–2012. Colonoscopy detected 512 CRC patients, 319 colonic cancer and 193 rectal cancer. Extra colonic malignancies were detected in 177 patients, 689 had adenomas of which 399 were high‐risk, 1,342 had nonneoplastic bowell disease and 1,978 subjects had ‘clean’ colorectum. Univariable analysis demonstrated that all biomarkers were statistically significant. Multivariate logistic regression demonstrated that the blood‐based biomarkers in combination significantly predicted the endpoints. The reduced model resulted in the selection of CEA, hs‐CRP, CyFra21‐1 and Ferritin for the two endpoints; AUCs were 0.76 and 0.84, respectively. The postive predictive value at 90% sensitivity was 25% for endpoint 1 and the negative predictive value was 93%. For endpoint 2, the postive predictive value was 18% and the negative predictive value was 97%. Combinations of serological protein biomarkers provided a significant identification of subjects with high risk of the presence of colorectal neoplasia. The present set of biomarkers could become important adjunct in early detection of CRC.     

Targeted imaging of breast tumor progression and therapeutic response in a human uMUC‐1 expressing transgenic mouse model

The ability to monitor breast cancer initiation and progression on the molecular level would provide an effective tool for early diagnosis and therapy. In the present study, we focused on the underglycosylated MUC‐1 tumor antigen (uMUC‐1), which is directly linked to tumor progression from pre‐malignancy to advanced malignancy in breast cancer and has been identified as the independent predictor of local recurrence and tumor response to chemotherapy. We investigated whether changes in uMUC‐1 expression during tumor development and therapeutic intervention could be monitored non‐invasively using molecular imaging approach with the uMUC‐1‐specific contrast agent (MN‐EPPT) detectable by magnetic resonance and fluorescence optical imaging. This was done in mice that express human uMUC‐1 tumor antigen (MMT mice) and develop spontaneous mammary carcinoma in a stage‐wise fashion. After the injection of MN‐EPPT there was a significant reduction in average T2 relaxation times of the mammary fat pad between pre‐malignancy and cancer. In addition, T2 relaxation times were already altered at pre‐malignant state in these mice compared to non‐tumor bearing mice. This indicated that targeting uMUC‐1 could be useful for detecting pre‐malignant transformation in the mammary fat pad. We also probed changes in uMUC‐1 expression with MN‐EPPT during therapy with doxorubicin (Dox). We observed that tumor delta‐T2s were significantly reduced by treatment with Dox indicating lower accumulation of MN‐EPPT. This correlated with a lower level of MUC‐1 expression in the Dox‐treated tumors, as confirmed by immunoblotting. Our study could provide a very sensitive molecular imaging approach for monitoring tumor progression and therapeutic response.     

Molecular imaging of glucose uptake in oral neoplasia following topical application of fluorescently labeled deoxy‐glucose

The clinical value of assessing tumor glucose metabolism via F‐18 fluorodeoxyglucose (FDG) PET imaging in oncology is well established; however, the poor spatial resolution of PET is a significant limitation especially for early stage lesions. An alternative technology is optical molecular imaging, which allows for subcellular spatial resolution and can be effectively used with topical contrast agents for imaging epithelial derived cancers. The goal of this study was to evaluate the potential of optical molecular imaging of glucose metabolism to aid in early detection of oral neoplasia. Fluorescently labeled deoxyglucose (2‐NBDG (2‐[N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino]‐2‐deoxy‐D ‐glucose)) was applied topically to tissue phantoms, fresh oral biopsies (n = 32) and resected tumors specimens (n = 2). High‐resolution imaging results show that 2‐NBDG can be rapidly delivered to oral epithelium using topical application. In normal epithelium, the uptake of 2‐NBDG is limited to basal epithelial cells. In contrast, high‐grade dysplasia and cancers show uptake of 2‐NBDG in neoplastic cells throughout the lesion. Following 2‐NBDG labeling, the mean fluorescence intensity of neoplastic tissue averages 3.7 times higher than that of matched nonneoplastic oral biopsies in samples from 20 patients. Widefield fluorescence images of 8‐paired oral specimens were obtained pre and postlabeling with 2‐NBDG. Prior to labeling, neoplastic samples showed significantly lower autofluorescence than nonneoplastic samples. The fluorescence of neoplastic samples increased dramatically after labeling; the differential increase in fluorescence was on average 30 times higher in neoplastic samples than in normal samples. Topical application of 2‐NBDG can therefore provide image contrast in both widefield and high‐resolution fluorescence imaging modalities, highlighting its potential in early detection of oral neoplasia. © 2008 Wiley‐Liss, Inc.     

In vivo imaging of membrane type‐1 matrix metalloproteinase with a novel activatable near‐infrared fluorescence probe

Membrane type‐1 matrix metalloproteinase (MT1‐MMP) is a protease activating MMP‐2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1‐MMP would be useful for tumor diagnosis, we developed a novel near‐infrared (NIR) fluorescence probe that can be activated following interaction with MT1‐MMP in vivo. MT1‐hIC7L is an activatable fluorescence probe comprised of anti‐MT1‐MMP monoclonal antibodies conjugated to self‐assembling polymer micelles that encapsulate NIR dyes (IC7‐1, λ_em: 858 nm) at concentrations sufficient to cause fluorescence self‐quenching. In aqueous buffer, MT1‐hIC7L fluorescence was suppressed to background levels and increased approximately 35.5‐fold in the presence of detergent. Cellular uptake experiments revealed that in MT1‐MMP positive C6 glioma cells, MT1‐hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti‐MT1‐MMP monoclonal antibody. In MT1‐MMP negative MCF‐7 breast adenocarcinoma cells, both MT1‐hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1‐hIC7L was suppressed by pre‐treatment with an MT1‐MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor‐bearing mice showed that MT1‐hIC7L specifically visualized C6 tumors (tumor‐to‐background ratios: 3.8 ± 0.3 [MT1‐hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF‐7 tumors. Together, these results show that MT1‐hIC7L would be a potential activatable NIR probe for specifically detecting MT1‐MMP‐expressing tumors.     

Early recognition of lung cancer by integrin targeted imaging in K‐ras mouse model

Non‐small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin‐targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K‐ras mutation. We conducted ex vivo and fluorescence molecular tomography‐X‐ray computed tomography (FMT‐XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin‐targeted fluorescent signal significantly differed between wild type lung tissue and K‐ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3‐integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4‐week‐old animals could be noninvasively detected in vivo on the fluorescence channel of FMT‐XCT. Our findings demonstrated αvβ3‐integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K‐ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.     

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis‐associated colon cancer

Fluorescence tumor imaging using exogenous fluorescent tumor‐targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis‐associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate‐induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31‐immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX‐2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E₂ promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast‐enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis‐associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX‐2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.     

Occurrence and significance of tumor‐associated neutrophils in patients with colorectal cancer

Inflammatory cells are an essential component of the tumor microenvironment. Neutrophils have emerged as important players in the orchestration and effector phase of innate and adaptive immunity. The significance of tumor‐associated neutrophils (TAN) in colorectal cancer (CRC) has been the subject of conflicting reports and the present study was designed to set up a reliable methodology to assess TAN infiltration in CRC and to evaluate their clinical significance. CD66b and myeloperoxidase (MPO) were assessed as candidate neutrophil markers in CRC using immunohistochemistry. CD66b was found to be a reliable marker to identify TAN in CRC tissues, whereas MPO also identified a subset of CD68⁺ macrophages. CRC patients (n = 271) (Stages I–IV) were investigated retrospectively by computer‐assisted imaging on whole tumor sections. TAN density dramatically decreases in Stage IV patients as compared to Stage I–III. At Cox analysis, higher TAN density was associated with better prognosis. Importantly, multivariate analysis showed that prognostic significance of TAN can be influenced by clinical stage and 5‐fluorouracil(5‐FU)‐based chemotherapy. On separate analysis of Stage III patients (n = 178), TAN density had a dual clinical significance depending on the use of 5‐FU‐based chemotherapy. Unexpectedly, higher TAN density was associated with better response to 5‐FU‐based chemotherapy. Thus, TAN are an important component of the immune cell infiltrate in CRC and assessment of TAN infiltration may help identify patients likely to benefit from 5‐FU‐based chemotherapy. These results call for a reassessment of the role of neutrophils in cancer using rigorous quantitative methodology.     

Evaluating tumor metastatic potential by imaging intratumoral acidosis via pH‐activatable near‐infrared fluorescent probe

Metastasis accounts for the vast majority of cancer deaths. To minimize metastasis‐associated mortality, it is crucially important to evaluate the metastatic potential (M.P.), that is, defined as a tendency of a primary tumor to colonize a distant organ. Dysregulated pH in solid tumors, especially the acidification of extracellular pH (pH_e) promotes dormant metastasis by driving protease‐mediated digestion, disrupting cell‐matrix interaction and increasing migration of cancer cells. Therefore, imaging intratumoral acidosis creates a unique opportunity to evaluate the M.P. In this work, a novel pH activatable probe was developed, in which two near‐infrared (NIR) fluorophores were conjugated via a flexible and acid liable linkage. While the fluorescence of this probe is quenched due to intramolecular dimeric aggregate under neutral environment, the cleavage of pH liable linkage with the concomitant disruption of aggregates in acidic tumor microenvironment results in a remarkable fluorescence enhancement. This probe not only visualized the primary tumors with high target to background (T/B) signal ratio in vivo, but also revealed the correlation between the M.P. and acidosis distribution pattern in tumor. While the acidosis locate dispersedly at tumor periphery in highly metastatic tumor, it distribute more widely in lowly metastatic tumor and the acidification degree increases substantially from the margin to core areas. This pH activatable NIR fluorescent probe holds the potential to evaluate the M.P., monitor the therapeutic response and predict the prognosis by delineating acidosis in tumors.     

Fluorescence‐lifetime molecular imaging can detect invisible peritoneal ovarian tumors in bloody ascites

Blood contamination, such as bloody ascites or hemorrhages during surgery, is a potential hazard for clinical application of fluorescence imaging. In order to overcome this problem, we investigate if fluorescence‐lifetime imaging helps to overcome this problem. Samples were prepared at concentrations ranging 0.3–2.4 μm and mixed with 0–10% of blood. Fluorescence intensities and lifetimes of samples were measured using a time‐domain fluorescence imager. Ovarian cancer SHIN3 cells overexpressing the D‐galactose receptor were injected into the peritoneal cavity 2.5 weeks before the experiments. Galactosyl serum albumin‐rhodamine green (GSA‐RhodG), which bound to the D‐galactose receptor and was internalized thereafter, was administered intraperitoneally to peritoneal ovarian cancer‐bearing mice with various degrees of bloody ascites. In vitro study showed a linear correlation between fluorescence intensity and probe concentration (r² > 0.99), whereas the fluorescence lifetime was consistent (range, 3.33 ± 0.15–3.75 ± 0.04 ns). By adding 10% of blood to samples, fluorescence intensities decreased to <1%, while fluorescence lifetimes were consistent. In vivo fluorescence lifetime of GSA‐RhodG stained tumors was longer than the autofluorescence lifetime (threshold, 2.87 ns). Tumor lesions under hemorrhagic peritonitis were not depicted using fluorescence intensity imaging; however, fluorescence‐lifetime imaging clearly detected tumor lesions by prolonged lifetimes. In conclusion, fluorescence‐lifetime imaging with GSA‐RhodG depicted ovarian cancer lesions, which were invisible in intensity images, in hemorrhagic ascites.     

Mesenchymal stromal cells induce epithelial‐to‐mesenchymal transition in human colorectal cancer cells through the expression of surface‐bound TGF‐β

Mesenchymal stem/stromal cells (MSC) are multipotent precursors endowed with the ability to home to primary and metastatic tumor sites, where they can integrate into the tumor‐associated stroma. However, molecular mechanisms and outcome of their interaction with cancer cells have not been fully clarified. In this study, we investigated the effects mediated by bone marrow‐derived MSC on human colorectal cancer (CRC) cells in vitro and in vivo. We found that MSC triggered epithelial‐to‐mesenchymal transition (EMT) in tumor cells in vitro, as indicated by upregulation of EMT‐related genes, downregulation of E‐cadherin and acquisition of mesenchymal morphology. These effects required cell‐to‐cell contact and were mediated by surface‐bound TGF‐β newly expressed on MSC upon coculture with tumor cells. In vivo tumor masses formed by MSC‐conditioned CRC cells were larger and characterized by higher vessel density, decreased E‐cadherin expression and increased expression of mesenchymal markers. Furthermore, MSC‐conditioned tumor cells displayed increased invasiveness in vitro and enhanced capacity to invade peripheral tissues in vivo. Thus, by promoting EMT‐related phenomena, MSC appear to favor the acquisition of an aggressive phenotype by CRC cells.     

Permissive expansion and homing of adoptively transferred T cells in tumor‐bearing hosts

Activated T cells expressing endogenous or transduced TCRs are two cell types currently used in clinical adoptive T‐cell therapy. The ability of these cells to recognize their antigen, expand and traffic to the tumor site are the initial steps necessary for successful therapy. In this study, we used in vivo bioluminescent imaging (BLI) of Renilla luciferase (RLuc) expressing T cells to evaluate the ability of adoptively transferred T cells to survive, expand and home to tumor site in vivo. Using this method, termed RT‐Rack (Rluc T cell tracking), we followed T‐cell response against tumors in vivo. Expansion and homing of adoptively transferred T cells were antigen dependent, but independent of the host immune status. Moreover, we successfully detected T‐cell response to small and large tumors, including autochthonous liver tumors. The adoptively transferred T cells were not ignorant or excluded in a partially tolerant host, which expressed low level of the target in the periphery. Using T cell receptor (TCR)‐engineered T cells, we showed the ability of these cells to respond in tumor‐bearing hosts by expanding and homing to the tumor site. In all these models, the host immune status, the nature of the tumor or of the antigen, the tumor size and the presence of the targeted antigen in the periphery did not prevent the adoptively transferred T cells from responding by expanding and homing to the tumor. However, T cells had higher expression of the inhibitory receptor PD1 and reduced functional activity when a self‐antigen was targeted.     

Time‐domain in vivo near infrared fluorescence imaging for evaluation of matriptase as a potential target for the development of novel,inhibitor‐based tumor therapies

Proteolytic enzymes expressed on the surface of tumor cells, and thus easily accessible to external interventions, represent useful targets for anticancer and antimetastatic therapies. In our study, we thoroughly evaluated matriptase, a trypsin‐like transmembrane serine protease, as potential target for novel inhibitor‐based tumor therapies. We applied time‐domain near infrared fluorescence (NIRF) imaging to characterize expression and activity of matriptase in vivo in an orthotopic AsPC‐1 pancreatic tumor model in nude mice. We show strong and tumor‐specific binding of intravenously injected Cy5.5 labeled antimatriptase antibody (MT‐Ab*Cy5.5) only to primary AsPC‐1 tumors and their metastases over time within living mice, taking into account fluorescence intensities and fluorescence lifetimes of the applied probes. Specific binding of MT‐Ab*Cy5.5 to tumor sites was confirmed by ex vivo NIRF imaging of tumor tissue, NIRF microscopy and by coregistration of the in vivo acquired NIRF intensity maps to anatomical structures visualized by flat‐panel volume computed tomography (fpVCT) in living mice. Moreover, using an activatable synthetic substrate S*DY‐681 we could clearly demonstrate that matriptase is proteolytically active in vitro as well as in vivo in tumor‐bearing mice, and that application of synthetic active‐site inhibitors having high affinity and selectivity toward matriptase can efficiently inhibit its proteolytic activity for at least 24 hr. We thus successfully applied NIRF imaging in combination with fpVCT to characterize matriptase as a promising molecular target for inhibitor‐based cancer therapies.     

Inhibition of MK2 suppresses IL‐1β, IL‐6, and TNF‐α‐dependent colorectal cancer growth

Colorectal cancer (CRC) development and progression is associated with chronic inflammation. We have identified the MAPK‐activated protein kinase 2 (MK2) pathway as a primary mediator of inflammation in CRC. MK2 signaling promotes production of proinflammatory cytokines IL‐1β, IL‐6 and TNF‐α. These cytokines have been implicated in tumor growth, invasion and metastasis. For the first time, we investigate whether MK2 inhibition can improve outcome in two mouse models of CRC. In our azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis‐associated CRC, MK2 inhibitor treatment eliminated murine tumor development. Using the implanted, syngeneic murine CRC cell line CT26, we observe significant tumor volume reduction following MK2 inhibition. Tumor cells treated with MK2 inhibitors produced 80% less IL‐1β, IL‐6 and TNF‐α and demonstrated decreased invasion. Replenishment of downstream proinflammatory MK2‐mediated cytokines (IL‐1β, IL‐6 and TNF‐α) to tumors led to restoration of tumor proliferation and rapid tumor regrowth. These results demonstrate the importance of MK2 in driving proinflammatory cytokine production, its relevance to in vivo tumor proliferation and invasion. Inhibition of MK2 may represent an attractive therapeutic target to suppress tumor growth and progression in patients.     

CHFR silencing or microsatellite instability is associated with increased antitumor activity of docetaxel or gemcitabine in colorectal cancer

Phenotypic differences among cancers with the same origin may be associated with chemotherapy response. CHFR silencing associated with DNA methylation has been suggested to be predictive of taxane sensitivity in diverse tumor types. However, the use of microsatellite instability (MSI:unstable–MSS:stable) as a predictive marker for therapeutic effect has had conflicting results. We examined these molecular alterations as predictors of chemotherapy sensitivity in colorectal cancer (CRC). Differential sensitivity to docetaxel and gemcitabine was compared to potential predictive biomarkers CHFR methylation and MSI status. Cell lines that were MSI‐H/CHFR‐methylated, MSS/CHFR‐methylated and MSS/CHFR‐unmethylated were assessed for in vivo sensitivity of CRC cell line xenografts to docetaxel and/or gemcitabine. We observed increased sensitivity in vitro to gemcitabine in cell lines with MSI and docetaxel in cell lines with CHFR inactivation via DNA methylation. In vivo treatment of human xenografts confirmed differential sensitivity, with the MSI‐H/CHFR‐methylated line RKO having tumor growth inhibition to each agent, and at least additive tumor growth inhibition with combination therapy. The MSS‐CHFR‐unmethylated line, CACO₂, was resistant to single and combination therapy, while COLO205, the MSS/CHFR‐methylated line, showed tumor growth inhibition with docetaxel, but not gemcitabine, therapy. CHFR methylation in CRC cell lines predicted for sensitivity in vitro and in vivo to docetaxel, while MSI‐H cell lines were more sensitive to gemcitabine. These data suggest that a subset of CRC patients would be selectively sensitive to a novel combination of gemcitabine and docetaxel, and are the basis for an ongoing clinical trial of this combination in a biomarker‐selected patient population.