Bioluminescence Imaging Imaging Research Articles

Development of an integrated dual-modality 3D bioluminescence tomography and ultrasound imaging system for small animal tumor imaging.

Ultrasound (US) is a valuable tool for imaging soft tissue and visualizing tumor contours. Taking the benefits of US, we presented an integrated dual-modality imaging system in this paper that achieves three-dimensional (3D) bioluminescence tomography (BLT) with multi-view bioluminescence images and 3D US imaging. The purpose of this system is to perform non-invasive, long-term monitoring of tumor growth in 3D images. US images can enhance the accuracy of the 3D BLT reconstruction and the bioluminescence dose within an object. Furthermore, an integrated co-registered scanning geometry was used to capture the fused BLT and US images. We validated the system with an in vivo experiment involving tumor-bearing mice. The results demonstrated the feasibility of reconstructing 3D BLT images in the tumor region using 3D US images. We used the dice coefficient and locational error to evaluate the similarity between the reconstructed source region and the actual source region. The dice coefficient was 88.5%, and the locational error was 0.4 mm when comparing the BLT and 3D US images. The hybrid BLT/US system could provide significant benefits for reconstructing the source of tumor location and conducting quantitative analysis of tumor size.

Abstract 3582: Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model

Abstract Purpose: There remains an unmet need to provide effective treatment therapy for patients with primary and metastatic brain tumors; lack of drug penetration across the blood brain barrier (BBB) is a key factor. Synthetic lethality remains an attractive mechanism in treating brain tumors post radiotherapy. Here, we evaluated brain penetration and distribution of niraparib and olaparib in a mouse brain tumor model. Experimental: Female mice (CrTac:NCr-Foxn1nu; 6 w/o) received 2.5E5 luciferase transfected human breast cancer line (MDA 231-BRM2-831) via intracardiac injection. Mice were imaged twice/week using bioluminescence imaging (BLI) to monitor tumor growth. On day 35, mice with brain metastases (BM) were treated via oral gavage once daily for 5 days with either niraparib (35 mg/kg, n=4 BM, n=3 control), olaparib (50 mg/kg, n=3 BM, n=3 control), or vehicle (n=3 control). Terminal blood samples and brains were collected 2 hr post final dose. Serial tissue sections were collected for MALDI-IMS, H&E and IHC staining from 5 distinct horizontal planes in the brain. Tissue collected between each imaging plane was homogenized for LC-MS bioanalysis. Summary of Data: In vivo BLI imaging was used to identify mice with BM and tumor presence was confirmed ex vivo using IHC. Quantitative MALDI IMS of coronal brain sections collected from mice administered niraparib showed consistent concentrations distributed throughout the brain parenchyma with locally higher concentrations detected from tumor regions. Table 1 summarizes the LC-MS bioanalysis concentrations in plasma and bulk brain homogenates and brain section concentrations detected by MALDI IMS. The estimated mean unbound brain-to-plasma partition coefficient (Kp,uu,brain) was 3.0x and 5.6x higher for niraparib compared to olaparib in control and BM mice, respectively. Conclusions: Herein, we demonstrated that niraparib has a higher brain penetration and distribution compared to olaparib in both control mice and mice with BM. Summary of Quantification Results Displayed as Total Conc. with Adjusted Free Conc. in Parentheses Compound Group Terminal Plasma Mean (ng/mL) LC-MS Bulk Brain Homogenate Mean (ng/g) MALDI IMS Brain Section Mean (ng/g) Kp,uu Brain Niraparib Control 3847 (769) 658 (111) 532 (89) 0.18 Niraparib BM 2535 (507) 543 (91) 473 (80) 0.14 Niraparib Vehicle BLQ BLQ Olaparib Control 226 (68) 6 (2) BLQ 0.06 Olaparib BM 119 (36) 5 (2) BLQ 0.03 Olaparib Vehicle BLQ BLQ Citation Format: Mark Reid Groseclose, Jeremy Barry, Tinamarie Skedzielewski, Gerald McDermott, Chakravarthi Balabhadrapatruni, Bill Benson, Yongle Pang, David K. Lim, Hoang Tran, Mike Ringenberg, Keyur Gada, Amine Aziez, Elaine Paul, Hasan Alsaid. Differentiation of niraparib and olaparib brain penetration in a mouse brain metastatic tumor model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3582.

Near-Infrared Bioluminescence Imaging of Macrophage Sensors for Cancer Detection In Vivo.

Melanoma is an aggressive type of skin cancer with a poor prognosis after it gets metastasized. The early detection of malignant melanoma is critical for effective therapy. Because melanoma often resembles moles, routine skin check-up may help for timely identification of suspicious areas. Recently, it has been shown that the interplay of melanoma cells with the immune system can help develop efficient therapeutic strategies. Here, we leveraged engineered macrophages (BMC2) as cell-based sensors for metastatic melanoma. To perform dual-color bioluminescence imaging (BLI) in vivo, macrophages were engineered to express a green click beetle luciferase (CBG2) and a near-infrared fluorescent dye (DiR), and B16F10 melanoma cells were instead engineered to express a near-infrared click beetle luciferase (CBR2). Using real-time in vivo dual-color BLI and near-infrared fluorescence (FL) imaging, we could demonstrate that macrophages were able to sense and substantially accumulate in subcutaneous and metastatic melanoma tissues at 72 h after systemic injections. Together, we showed the potentiality to use optical imaging technologies to track circulating macrophages for the non-invasive detection of metastatic melanoma.

Mature Osteoblasts Are Critical Bone Marrow Niche Cells Regulating Multiple Myeloma Cell Growth, at Least in Part, By Affecting the Local Immune Microenvironment

Despite high response rates to new therapies, most multiple myeloma (MM) patients relapse and become refractory to treatment. This is likely because MM cells enter dormancy and reactivate only under permissive conditions. Cellular dormancy is ubiquitous in normal tissues as well as in malignant, non-malignant, and metastasizing tumors. Dormant tumor cells (DTC) are protected from therapies which can reactivate later at the time of disease progression, resulting in relapsed refractory MM (RRMM). The tumor microenvironment (TME) is a key determinant of tumor dormancy and reactivation. We have previously shown that inhibition of Activin A in SCID mice transplanted with human MM.1S increased osteoblast numbers and resulted in inhibition of MM growth (Vallet et al., PNAS). We hypothesize that MM-DTCs reside in osteoblastic niche in the BM TME. Our long-term goal is to identify factors regulating DTCs in MM and develop strategies for long term disease control.We have generated mice in which mature osteoblasts can be postnatally deleted in an inducible and reversible manner. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). Until the treatment with diphtheria toxin (DT), the OC-Cre/iDTR mice were immunocompetent compared to iDTR mice, as they did not show any differences in immune cell populations by flow cytometry, and did not show any skeletal phenotype, as measured by micro-computed CT (micro-CT).We chose to induce mature osteoblast-deficiency at 8-weeks of age to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x10 6 5TGM1 luciferase tdTomato positive (5TGM1-Luc-Tom) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-Tom cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-Tom cells at 4-weeks showed dramatic increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-Tom cells in other long-bones of OC-Cre/iDTR mice but not the controls. To identify local BM TME changes, we assessed the levels of 200 cytokines and growth factors in the BM from these mice. The levels of immune regulatory cytokines, such as IL-17F and IL-21, were found to be decreased in the OC-Cre/iDTR mice compared to control iDTR mice, both after 4-weeks from intratibial 5TGM1 cell injection. This was accompanied by significant decrease in the fraction of CD4+ T-helper and Th17+ cells in the OC-Cre/iDTR mice compared to controls, as assessed by flow cytometry.These data show that MM cells engraft and proliferate rapidly within the BM in the absence of mature osteoblasts and have a propensity to migrate to other long bones with time. This may be partially mediated by the altered immune cell profile in the BM in the absence of mature osteoblasts. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden creating an environment for long term tumor control. Importantly, this model will allow us to understand the osteoblastic niche for MM and the mechanisms of activation and dormancy, at least in part, via a role of the immune cells. DisclosuresFulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

CRISPR/Cas9-edited triple-fusion reporter gene imaging of dynamics and function of transplanted human urinary-induced pluripotent stem cell-derived cardiomyocytes.

To investigate the post-transplantation behaviour and therapeutic efficacy of human urinary-induced pluripotent stem cell-derived cardiomyocytes (hUiCMs) in infarcted heart. We used clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology to integrate a triple-fusion (TF) reporter gene into the AAVS1 locus in human urine-derived hiPSCs (hUiPSCs) to generate TF-hUiPSCs that stably expressed monomeric red fluorescent protein for fluorescence imaging, firefly luciferase for bioluminescence imaging (BLI) and herpes simplex virus thymidine kinase for positron emission tomography (PET) imaging. Transplanted cardiomyocytes derived from TF-hUiPSCs (TF-hUiCMs) engrafted and proliferated in the infarcted heart as monitored by both BLI and PET imaging and significantly improved cardiac function. Under ischaemic conditions, TF-hUiCMs enhanced cardiomyocyte (CM) glucose metabolism and promoted angiogenic activity. This study established a CRISPR/Cas9-mediated multimodality reporter gene imaging system that can determine the dynamics and function of TF-hUiCMs in myocardial infarction, which is helpful for investigating the application of stem cell therapy.

A Novel in-Vivo Model to Examine Homing of Multiple Myeloma Cells in Postnatal, Inducible, and Reversible Loss of Mature Osteoblasts

The initiation of Multiple myeloma (MM) coincides with a decrease in osteoblasts and an increase in osteoclasts and adipocytes. However, the contribution of osteoblasts to the initiation and progression of MM remains mostly unknown. In-vitro studies have shown that co-cultures with MC3T3 murine osteoblast cell-line induced quiescence in 5TGM1 murine myeloma cells. In SCID or C57BL/KaLwRijHsd mice, the fluorescently labeled MM cell lines, injected through tail-vein, co-localized with endosteal osteoblasts and were found to be quiescent. We previously demonstrated that increased osteoblast numbers using Activin A inhibition in SCID mice with human-origin MM.1S cells resulted in inhibition of MM growth (Vallet et al., PNAS 2010). Here we aimed to develop a genetic in-vivo model for a better understanding of the interactions between osteoblasts and MM cells. Osteolineage cells develop through the stages of pre-osteoblasts, committed osteoblasts, mature osteoblasts, and osteocytes; each stage governed by a distinct molecular signature and function. To delineate these differences, individual osteoblast populations were FACS-sorted from long bones of Osterix-GFP+ (Osx+) mice (pre-osteoblasts), Collagen 2.3-GFP+ (Col2.3+) mice (committed osteoblasts), and Osteocalcin-YFP+ (OCN+) mice (mature osteoblasts). Co-cultured of these osteoblast populations with 5TGM1 MM cells showed that the OCN+ mature osteoblasts suppressed MM proliferation the most. Therefore, we focused our studies on the role of mature osteoblasts in MM homing and engraftment. We next generated mice in which mature osteoblasts were postnatally deleted in an inducible and reversible manner to better understand the molecular mechanisms of engraftment, proliferation, and migration of 5TGM1 injected intra-tibialy. To achieve this, mice carrying floxed diphtheria toxin receptor (DTR) alleles were mated with mice expressing Cre-recombinase driven by the osteocalcin promoter (OC-Cre). This led to the expression of DTR in mature osteoblasts (OC-Cre/iDTR) only. The control mice were littermates lacking the OC-Cre allele (iDTR). The OC-Cre/iDTR mice were indistinguishable from the controls until treated with diphtheria toxin (DT). We chose to induce mature osteoblast-deficiency at 8-weeks to allow skeletal maturation which is assumed to be completed by 6-7weeks of age. To induce postnatal deletion of mature osteoblasts, the OC-Cre/iDTR and control mice both were treated with 50 µg/Kg DT once a week, beginning at 8-weeks of age. Micro-CT analysis showed a significant increase in cortical porosity within 1-week after DT injection. 8-weeks of DT treatment significantly reduced trabecular bone fraction (BV/TV), trabecular numbers (Tb.N), and bone mineral density (BMD) with a significant increase in trabecular spacing (Tb.Sp). Immunohistochemistry for osteocalcin showed rapid loss of mature and endosteal osteoblasts (N.Ob/T.Ar). This was accompanied with a marked decreased in serum sclerostin and serum osteocalcin levels suggesting reduced osteocytes and mature osteoblasts, respectively. Importantly, serum CTX levels or numbers of osteoclasts (N.Oc/T.Ar) were unchanged. To study MM engraftment and progression, 3x106 5TGM1 luciferase eGFP positive (5TGM1-Luc-GFP) MM cells were injected into the tibia of OC-Cre/iDTR and control iDTR mice followed by weekly injection of DT for 8-weeks. Flow cytometry analysis showed a 4-fold increase in the 5TGM1-GFP cells in the BM from OC-Cre/iDTR mice compared to controls. Bioluminescence imaging (BLI) for 5TGM1-Luc-GFP cells at 4-weeks showed 600-fold increase in the OC-Cre/iDTR mice compared to controls. Interestingly, by 8-weeks, the BLI imaging showed 5TGM1-Luc-GFP cells in other long-bones of OC-Cre/iDTR mice but not the controls. These data show that MM cells engraft and proliferate rapidly in the absence of mature osteoblasts in-vivo. Moreover, in the absence of mature osteoblasts, MM cells have a propensity to migrate to other long bones. These data further suggest that expanding the mature osteoblast niche may provide novel therapeutic avenues and reduce disease burden and create an environment for long term tumor control. Importantly, this model will allow us to follow and analyze the sequential engraftment, dormancy, reactivation, proliferation, and migration of myeloma cells, and evaluate the effects of osteoanabolic and anti-myeloma therapies. Disclosures Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:BMS: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Astrazeneca: Consultancy; Bluebird, Bio: Consultancy, Research Funding; Takeda: Consultancy; Immuneel: Membership on an entity's Board of Directors or advisory committees; Caribou: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

In Vivo Bioluminescence Imaging for Targeting Acute Hypoxic/Ischemic Small Intestine with Engineered Salmonella typhimurium

This study aimed at investigating the feasibility of bioluminescence imaging (BLI) with engineered Salmonella typhimurium (ΔppGpp S. typhimurium) for visualizing acute hypoxic/ischemic bowels. At the start of 12- or 24-h reperfusion, ΔppGpp S. typhimurium was injected into the lateral tail veins of rats in which three segments of the small intestine were respectively subjected to 2, 3, and 4 h of ischemia. BLI and magnetic resonance imaging were performed at each reperfusion time point. Bioluminescence was exclusively detected in the hypoxic/ischemic segment of the intestine, showing the ability of ΔppGpp S. typhimurium to specifically target and proliferate in a hypoxic/ischemic area. Serial monitoring of these rat models revealed a progressive increase in bacterial bioluminescence in the ischemic intestines in conjunction with viable bacterial counts. The viable bacterial counts were positively correlated with lactate dehydrogenase levels after 24 h of reperfusion following 3 or 4 h of ischemia as well as interleukin-6 levels after 24 h of reperfusion following 4 h of ischemia. Our findings demonstrated that BLI was able to detect the acute hypoxic/ischemic bowel via monitoring of the distribution, internalization, and activity of administered ΔppGpp S. typhimurium. These findings may be useful for the early diagnosis of ischemic bowel disease.

In vivo bioluminescence tomography-guided radiation research platform for pancreatic cancer: an initial study using subcutaneous and orthotopic pancreatic tumor models.

Genetically engineered mouse model(GEMM) that develops pancreatic ductal adenocarcinoma(PDAC) offers an experimental system to advance our understanding of radiotherapy(RT) for pancreatic cancer. Cone beam CT(CBCT)-guided small animal radiation research platform(SARRP) has been developed to mimic the RT used for human. However, we recognized that CBCT is inadequate to localize the PDAC growing in low image contrast environment. We innovated bioluminescence tomography(BLT) to guide SARRP irradiation for in vivo PDAC. Before working on the complex PDAC-GEMM, we first validated our BLT target localization using subcutaneous and orthotopic pancreatic tumor models. Our BLT process involves the animal transport between the BLT system and SARRP. We inserted a titanium wire into the orthotopic tumor as the fiducial marker to track the tumor location and to validate the BLT reconstruction accuracy. Our data shows that with careful animal handling, minimum disturbance for target position was introduced during our BLT imaging procedure(<0.5mm). However, from longitudinal 2D bioluminescence image(BLI) study, the day-to-day location variation for an abdominal tumor can be significant. We also showed that the 2D BLI in single projection setting cannot accurately capture the abdominal tumor location. It renders that 3D BLT with multiple-projection is needed to quantify the tumor volume and location for precise radiation research. Our initial results show the BLT can retrieve the location at 2mm accuracy for both tumor models, and the tumor volume can be delineated within 25% accuracy. The study for the subcutaneous and orthotopic models will provide us valuable knowledge for BLT-guided PDAC-GEMM radiation research.

Magnetic Resonance Imaging and Bioluminescence Imaging for Evaluating Tumor Burden in Orthotopic Colon Cancer

Quantifying tumor burden is important for following the natural history of orthotopic colon cancer and therapeutic efficacy. Bioluminescence imaging (BLI) is commonly used for such assessment and has both advantages and limitations. We compared BLI and magnetic resonance imaging (MRI) for quantifying orthotopic tumors in a mouse model of colon cancer. Among sequences tested, T2-based MRI imaging ranked best overall for colon cancer border delineation, contrast, and conspicuity. Longitudinal MRI detected tumor outside the colon, indistinguished by BLI. Colon tumor weights calculated from MRI in vivo correlated highly with tumor weights measured ex vivo whereas the BLI signal intensities correlated relatively poorly and this difference in correlations was highly significant. This suggests that MRI may more accurately assess tumor burden in longitudinal monitoring of orthotopic colon cancer in this model as well as in other models.

An orthotopic non-small cell lung cancer model for image-guided small animal radiotherapy platforms.

An orthotopic non-small cell lung cancer model in NMRI-nude mice was established to investigate the complementary information acquired from 80 kVp microcone-beam CT (micro-CBCT) and bioluminescence imaging (BLI) using different angles and filter settings. Different micro-CBCT-based radiation-delivery plans were evaluated based on their dose-volume histogram metrics of tumor and organs at risk to select the optimal treatment plan. H1299 cell suspensions injected directly into the lung render exponentially growing single tumor nodules whose CBCT-based volume quantification strongly correlated with BLI-integrated intensity. Parallel-opposed single angle beam plans through a single lung are preferred for smaller tumors, whereas for larger tumors, plans that spread the radiation dose across healthy tissues are favored. Closely mimicking a clinical setting for lung cancer with highly advanced preclinical radiation treatment planning is possible in mice developing orthotopic lung tumors. BLI and CBCT imaging of orthotopic lung tumors provide complementary information in a temporal manner. The optimal radiotherapy plan is tumor volume-dependent.

PO-022 Benzamide class of HDACi differentially enhance sodium iodide symporter expression in breast cancer

IntroductionSodium iodide symporter (hNIS) expression is reported across all subtypes of breast cancer (BC) and is a potential endogenous gene target for radioiodine therapy [PlosOne8(1:e54055, 2012]. Successful clinical application however relies on very high expression of functional NIS in BC cells. We have been exploiting various transcriptional regulation approaches for NIS, of which use of HDAC inhibitors (HDACi) have shown promising amplification of endogenous NIS expression in BC [Scientific Reports6:19341, 2016]. Here, we show that differential induction of NIS expression in BC is possible using Benzamide class of HDACi (bHDACi) with minimal off-target effects on other cell types.Material and methodsThe effect of bHDACi, i.e. CI-994, Chidamide, MS-275 and AR-42, on NIS gene transcription was verified on a panel of human cancer cells by RTPCR. Functional in vitro assessment was done by non-radioactive iodine uptake. I131therapeutic efficacy was measured by clonogenic assay. Promoter binding transcription factor (TF) profiler array was done to identify novel TFs responsible for differential activation in BC cells. Further, in vivo therapeutic efficacy was monitored by bioluminescence imaging (BLI) of ZR-75–1-fl2.tdt orthotopic model.Results and discussionsUsing a non-toxic, low dose of bHDACi(s) treatment on a cancer cell panel, including breast, thyroid, lung, glioma, fibrosarcoma and ovarian, a significant induction of NIS expression (p<0.05) was observed only in BC cells. Observed differences of NIS expression was confirmed by protein expression and functional assessment on BC cells. Further, novel TFs (FOXA1, FOXOF2 etc.) were identified which show differential activation in BC after CI-994 treatment. Only in case of BC cells pre-treated with bHDACi(s), cell survival assay results showed enhanced cell death on exposure to I131. Further, AR-42 drug was tested in vivo which showed similar results. AR-42 pre-treated ZR-75–1 fl2tdt orthotopic tumour showed increased NIS expression. In comparison to untreated and only I131 treated group, a combination treatment of AR-42 with 1mCi I131, BLI imaging showed 20% and 30% signal drop on day1 and day 2 respectively indicating specific radio-ablation effect.ConclusionbHDACi(s) show differentially higher regulation of endogenous NIS with minimal effect on non-breast tissues and thus may potentially benefit NIS-based radioablation therapy trials in BC patients with low/moderate expression of NIS.

Migration of mesenchymal stem cells to tumor xenograft models and in vitro drug delivery by doxorubicin.

Mesenchymal stem cells (MSCs) show therapeutic effects in various types of diseases. MSCs have been shown to migrate towards inflamed or cancerous tissues, and visualized after sacrificing the animal. MSCs are able to deliver drugs to target cells, and are an ideal candidate for cancer therapy. The purpose of this study was to track the migration of MSCs in tumor-bearing mice; MSCs were also used as drug delivery vehicles. Human breast cancer cells (MDA-MB-231) and anaplastic thyroid cancer cells (CAL62) were transduced with lentiviral particles, to express the Renilla luciferase and mCherry (mCherry-Rluc) reporter genes. Human bone marrow-derived MSCs were transduced with lentiviral particles, to express the firefly luciferase and enhanced green fluorescence protein (Fluc2-eGFP) reporter genes (MSC/Fluc). Luciferase activity of the transduced cells was measured by bioluminescence imaging (BLI). Further in vitro migration assays were performed to confirm cancer cells conditioned medium dependent MSC and doxorubicin (DOX) treated MSC migration. MSCs were loaded with DOX, and their therapeutic effects against the cancer cells were studied in vitro. In vivo MSC/Fluc migration in mice having thyroid or breast cancer xenografts was evaluated after systemic injection. Rluc activity of CAL62/Rluc (R2=0.911), MDA-MB-231/Rluc (R2=0.934) cells and Fluc activity of MSC/Fluc (R2=0.91) cells increased with increasing cell numbers, as seen by BLI. eGFP expression of MSC/Fluc was confirmed by confocal microscopy. Similar migration potential was observed between MSC/Fluc and naïve MSCs in migration assay. DOX treated MSCs migration was not decreased compared than MSCs. Migration of the systemically injected MSC/Fluc cells into tumor xenografts (thyroid and breast cancer) was visualized in animal models (p<0.05) and confirmed by ex vivo (p<0.05) BLI. Additionally, MSCs delivered DOX to CAL62/Rluc and MDA-MB-231/Rluc cells, thereby decreasing their Rluc activities. In this study, we confirmed the migration of MSCs to tumor sites in cancer xenograft models using both in vivo and ex vivo BLI imaging. DOX-pretreated MSCs showed enhanced cytotoxic effects. Therefore, this noninvasive reporter gene (Fluc2)-based BLI may be useful for visualizing in vivo tracking of MSCs, which can be used as a drug delivery vehicle for cancer therapy.

Abstract 875: Improved resection and prolonged overall survival with PD-1-IRDye800CW fluorescent probe-guided surgery and PD-1 adjuvant immunotherapy

Abstract Purpose: An intraoperative technique to accurately identify microscopic tumor residuals could be applied to decrease the risk of positive surgical margins. Several lines of evidence support the expression and immunotherapeutic effect of PD-1 in breast cancer. Here, we sought to develop a fluorescence labeled PD-1 probe for in vivo breast tumor imaging and image-guided surgery. The efficacy of PD-1 monoclonal antibody (mAb) as adjuvant immunotherapy after surgery was also assessed. Experimental Design: A near-infrared dye labeled PD-1-IRDye800CW probe was developed and examined for its application in tumor imaging and image-guided tumor resection in an immunocompetent 4T1 mammary mouse tumor model that recapitulates clinical metastatic cancer. Fluorescence molecular imaging (FMI) was performed to monitor probe biodistribution and intraoperative imaging. Bioluminescence imaging (BLI) was carried out concurrently to monitor tumor growth and evaluate post-surgical tumor residuals, recurrences, and metastases. Results: The PD-1-IRDye800CW probe exhibited a specific and stable signal at the tumor region compared to the IgG control, and exhibited the highest tumor-to-background ratio (TBR) 8 h post-injection (11.62 ± 0.79), approximately 5 fold higher than the IgG control (2.13 ± 0.30). Furthermore, PD-1-IRDye800CW guided surgery combined with PD-1 adjuvant immunotherapy inhibited tumor regrowth and microtumor metastases, as determined by BLI imaging, and thus improved survival rate with no obvious side effects. Conclusions: Our study demonstrated the feasibility of using PD-1-IRDye800CW for breast tumor imaging and image-guided tumor resection. Moreover, we found that PD-1 mAb adjuvant immunotherapy reduces cancer recurrences and metastases emanating from minimal tumor residuals. Citation Format: Yang Du, Jie Tian. Improved resection and prolonged overall survival with PD-1-IRDye800CW fluorescent probe-guided surgery and PD-1 adjuvant immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 875. doi:10.1158/1538-7445.AM2017-875

Abstract 2660: PD-1 blockade in combination with zoledronic acid to enhance the antitumor efficacy in the breast cancer mouse model

Abstract Purpose: Blockade of PD-1 receptor may provide proof of concepts for the activity of an immune-modulation approach for the treatment of a breast cancer (BC). Zoledronic acid (ZA) has been proven to inhibit angiogenesis, invasion, and adhesion of tumor cells. The aim of this study was to investigate the potential of antibody treatment towards the T-cell checkpoint inhibitor PD-1 alone or in combination with ZA in BC mouse model. Experimental Design: The 4T1-Luc mouse mammary tumor model was used in this study. The anti-tumor efficacy of anti-PD-1 antibody alone or in combination with ZA was monitored by measuring tumor volume and bioluminescence imaging (BLI). At the end of study, the tumor immunohistochemistry was performed to confirm the in vivo observation and flow cytometry was used to determine the immune cell population in tumors. The survival rate was also calculated to evaluate the treatment efficacy. Results: The results showed that mice treated with the combination therapy exhibited the best antitumor response compared to untreated controls or single therapy. Furthermore, Combination therapy inhibited tumor regrowth as determined by BLI imaging, and thus significantly improved the median overall survival compared to other groups. Conclusions: Our study provides evidence for enhanced clinical BC treatment benefit from targeting co-signal molecules by combining PD-1 blockade with ZA treatment. Citation Format: Yuan Li, Yang Du, Ting Sun, Huadan Xue, Jie Tian, Zhengyu Jin. PD-1 blockade in combination with zoledronic acid to enhance the antitumor efficacy in the breast cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2660. doi:10.1158/1538-7445.AM2017-2660

Post-Transplant Cyclophosphamide Overcomes Graft-Versus-Host Disease Aggravation Induced By Extended Administration of Proteasome Inhibitors

The concept of suppressing dendritic cells (DCs) to prevent graft-versus-host disease (GvHD) following allogeneic hematopoietic stem cell transplantation (HSCT) has been rejuvenated since the introduction of proteasome inhibitors into clinical practice. Although early post-transplant administration of proteasome inhibitors ameliorates GvHD, delayed administration has been associated with alloreactivity-dependent gastrointestinal toxicity. This phenomenon is associated with a surge in interleukin-1β (IL-1β) and acceleration in donor T cell expansion. To address this issue, we sought to investigate whether in vivo depletion of donor T cells by concomitant administration of post-transplant cyclophosphamide (C) could overcome this phenomenon. Ten-week old BALB/c mice were irradiated (10 grays in two fractions) on day -1 and transplanted with 5x106 BM cells from C57BL6 mice. GvH inocula consisted of 5x106 splenocytes from C57BL6 (some post-CFSE labeling) or B6;FVB-PtprcaTg(CAG-Luc,-GFP)L2G85Chco Thy1a/J (ubiquitously transgenic for firefly luciferase for bioluminescence imaging (BLI). All mice were purchased from Jackson Laboratory and kept in a pathogen-free environment. Mice were untreated or received ixazomib (Z) (30μg, subcutaneously) on days -1, +2, and +5, C (1 mg intraperitoneally) on day +2, or both Z and C according to the same schedule. Mice were monitored for weight, GvHD score, and survival or sacrificed on day +6 for cytokine measurement and splenic cell counts. For BLI, mice receiving transgenic splenocytes were injected with 150mg/kg of D-Luciferin intraperitoneally and imaged 25 minutes later. GvHD worsening and sudden death occurred in a fraction of mice receiving Z following day +5 injection. The phenomenon was completely prevented by the addition of C. Overall, the group receiving both Z and C had significantly improved weight, GvHD score, and survival when compared to the mice left untreated or receiving either drug alone (figure 1). The addition of C to Z prevented IL-1β increase, was associated with suppression of tumor necrosis factor α (TNFα), and resulted in a profound depletion of splenic total and donor CD4+ cells. Furthermore, proliferating cells (CFSE low) were preferentially depleted as opposed to quiescent cells (CFSE high). BLI imaging showed an increase in signal intensity shortly after the administration of Z on day +5. On the other hand, the mice receiving C in addition to Z exhibited significantly lower donor T cell proliferation when compared to the other groups (figure 2).These findings suggest that C can prevent the phenomenon of GvHD acceleration associated with the extended administration of proteasome inhibitors, suppress cytokine production, and effectively reduce splenic total and donor CD4+ cell expansion with preferential depletion of proliferating as opposed to quiescent cells. The combination of Z and C seems a promising duplet for the prevention of GvHD. When Z is administered late following allogeneic HSCT for the prevention of disease relapse, the addition of C should be considered in order to avoid any unwanted aggravation of alloreactivity. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

In Vivo Follow-up of Brain Tumor Growth via Bioluminescence Imaging and Fluorescence Tomography.

Reporter gene-based strategies are widely used in experimental oncology. Bioluminescence imaging (BLI) using the firefly luciferase (Fluc) as a reporter gene and d-luciferin as a substrate is currently the most widely employed technique. The present paper compares the performances of BLI imaging with fluorescence imaging using the near infrared fluorescent protein (iRFP) to monitor brain tumor growth in mice. Fluorescence imaging includes fluorescence reflectance imaging (FRI), fluorescence diffuse optical tomography (fDOT), and fluorescence molecular Imaging (FMT®). A U87 cell line was genetically modified for constitutive expression of both the encoding Fluc and iRFP reporter genes and assayed for cell, subcutaneous tumor and brain tumor imaging. On cultured cells, BLI was more sensitive than FRI; in vivo, tumors were first detected by BLI. Fluorescence of iRFP provided convenient tools such as flux cytometry, direct detection of the fluorescent protein on histological slices, and fluorescent tomography that allowed for 3D localization and absolute quantification of the fluorescent signal in brain tumors.

Preclinical Activity of the Vascular Disrupting Agent OXi4503 against Head and Neck Cancer.

Vascular disrupting agents (VDAs) represent a relatively distinct class of agents that target established blood vessels in tumors. In this study, we examined the preclinical activity of the second-generation VDA OXi4503 against human head and neck squamous cell carcinoma (HNSCC). Studies were performed in subcutaneous and orthotopic FaDu-luc HNSCC xenografts established in immunodeficient mice. In the subcutaneous model, bioluminescence imaging (BLI) along with tumor growth measurements was performed to assess tumor response to therapy. In mice bearing orthotopic tumors, a dual modality imaging approach based on BLI and magnetic resonance imaging (MRI) was utilized. Correlative histologic assessment of tumors was performed to validate imaging data. Dynamic BLI revealed a marked reduction in radiance within a few hours of OXi4503 administration compared to baseline levels. However, this reduction was transient with vascular recovery observed at 24 h post treatment. A single injection of OXi4503 (40 mg/kg) resulted in a significant (p < 0.01) tumor growth inhibition of subcutaneous FaDu-luc xenografts. MRI revealed a significant reduction (p < 0.05) in volume of orthotopic tumors at 10 days post two doses of OXi4503 treatment. Corresponding histologic (H&E) sections of Oxi4503 treated tumors showed extensive areas of necrosis and hemorrhaging compared to untreated controls. To the best of our knowledge, this is the first report, on the activity of Oxi4503 against HNSCC. These results demonstrate the potential of tumor-VDAs in head and neck cancer. Further examination of the antivascular and antitumor activity of Oxi4503 against HNSCC alone and in combination with chemotherapy and radiation is warranted.

Route of delivery influences biodistribution of human bone marrow-derived mesenchymal stromal cells following experimental bone marrow transplantation

Mesenchymal stromal cells (MSCs) have shown promise as treatment for graft-versus-host disease (GvHD) following allogeneic bone marrow transplantation (alloBMT). Mechanisms mediating in vivo effects of MSCs remain largely unknown, including their biodistribution following infusion. To this end, human bone-marrow derived MSCs (hMSCs) were injected via carotid artery (IA) or tail vein (TV) into allogeneic and syngeneic BMT recipient mice. Following xenogeneic transplantation, MSC biodistribution was measured by bioluminescence imaging (BLI) using hMSCs transduced with a reporter gene system containing luciferase and by scintigraphic imaging using hMSCs labeled with [(99m)Tc]-HMPAO. Although hMSCs initially accumulated in the lungs in both transplant groups, more cells migrated to organs in alloBMT recipient as measured by in vivo BLI and scintigraphy and confirmed by ex vivo BLI imaging, immunohistochemistry and quantitative RT-PCR. IA injection resulted in persistent whole-body hMSC distribution in alloBMT recipients, while hMSCs were rapidly cleared in the syngeneic animals within one week. In contrast, TV-injected hMSCs were mainly seen in the lungs with fewer cells traveling to other organs. Summarily, these results demonstrate the potential use of IA injection to alter hMSC biodistribution in order to more effectively deliver hMSCs to targeted tissues and microenvironments.

Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

BackgroundHere we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.Methodology/Principal FindingsqPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.Conclusions/SignificanceThese data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

Development of a High-Throughput Molecular Imaging-Based Orthotopic Hepatocellular Carcinoma Model.

We have developed a novel orthotopic rat hepatocellular (HCC) model and have assessed the ability to use bioluminescence imaging (BLI), positron emission tomography (PET), and ultrasound for early tumor detection and monitoring of disease progression. Briefly, rat HCC cells were stably transfected with click beetle red as a reporter gene for BLI. Tumor cells were injected under direct visualization into the left or middle lobe of the liver in 37 rats. In six animals, serial PET, BLI, and ultrasound imaging were performed at 10-time points in 28 days. The remainder of the animals underwent PET imaging at 14 days. Tumor implantation was successful in 34 of 37 animals (91.9%). In the six animals that underwent serial imaging, tumor formation was first detected with BLI on Day 4 with continued increase through Day 21, and hypermetabolic activity on PET was first noted on Days 14-15 with continued increase through Day 28. PET activity was seen on Day 14 in the 28 other animals that demonstrated tumor development. Anatomic tumor formation was detected with ultrasound at Days 10-12 with continued growth through Day 28. The first metastases were detected by PET after Day 24. We have successfully developed and validated a novel orthotopic HCC small animal model that permits longitudinal assessment of change in tumor size using molecular imaging techniques. BLI is the most sensitive imaging method for detection of early tumor formation and growth. This model permits high-throughput in vivo evaluation of image-guided therapies.