Luciferase-expressing Tumor Cells Research Articles

Peritoneal carcinomatosis in mouse models.

Peritoneal carcinomatosis (PCa) represents a metastatic stage of a disease with unmet therapeutic options. Malignant cells from primary tumors (gastrointestinal or gynecologic malignancies) invade the peritoneal cavity and eventually seed onto peritoneal surfaces, with the omentum being the most common nest area. With a median survival of less than 6 months, PCa has a dismal prognosis that can be improved with treatments only available to a select few individuals with low tumor burden. Thus, the discovery of novel and effective therapies for this disease depends on reliable animal models. Here, we describe a method to generate syngeneic PCa mouse models based on intraperitoneal (i.p.) administration of tumor cells. This model allows to follow-up cancer progression in PCa models from ovarian and colorectal origins monitoring mice bodyweight changes, ascites development and overall survival. Moreover, luciferase-expressing tumor cells can also be used to assess tumor growth after i.p. injection through in vivo bioluminescence quantification. The establishment of reliable, easy-to-monitor and reproducible intraperitoneal syngeneic tumors models, as described here, is the first step to develop cutting-edge therapies against PCa.

Adoptive immunotherapy with cells from tumor-draining lymph nodes activated and expanded in vitro.

Tumor-draining lymph nodes (tumor-DLNs) provide a rich source of tumor-reactive lymphocytes which can be used in adoptive immunotherapy (AIT) and that circumvent the need to resect autologous tumor, without the challenges and shortcomings associated with using autologous tumor or anti-CD3 monoclonal antibody. Bryostatin/Ionomycin (Bryo/Io) provide a useful method of activating tumor-DLNs such that they can readily be expanded to sufficient numbers to be used in AIT, and growing the tumor-DLN lymphocytes in the gamma chain cytokines IL-7 plus IL-15 is superior to IL-2 in terms of T cell numbers and phenotype. AIT with these cells induces tumor regression and provides protection against metastases and future tumor challenge. Here, we provide a stepwise protocol to sensitize tumor-DLN cells in donor mice, activate tumor-DLN T cells ex vivo using Bryo/Io, expansion of these cells in gamma chain cytokines and adoptive transfer of the expanded cells back into tumor-bearing hosts. Methods relevant to these experiments, such as injecting tumor cells intravenously and monitoring for pulmonary metastases, tumor volume measurement and resection, and use of luciferase-expressing tumor cells to monitor for metastases following resection, are described in detail. The methods outlined herein can be easily adapted to suit similar experiments across multiple tumor cell lines and syngeneic mouse models.

Protocols for the Evaluation of a Lymphatic Drug Delivery System Combined with Bioluminescence to Treat Metastatic Lymph Nodes.

Bioluminescence (BL) imaging is a powerful non-invasive imaging modality widely used in a broad range of biological disciplines for many types of measurements. The applications of BL imaging in biomedicine are diverse, including tracking bacterial progression, research on gene expression patterns, monitoring tumor cell growth/regression or treatment responses, determining the location and proliferation of stem cells, and so on. It is particularly valuable when studying tissues at depths of 1 to 2cm in mouse models during preclinical research. Here we describe the protocols for the therapeutic evaluation of a lymphatic drug delivery system (LDDS) using an in vivo BL imaging system (IVIS) for the treatment of metastatic lymph nodes (LNs) with 5-fluorouracil (5-FU). The LDDS is a method that directly injects anticancer drugs into sentinel LNs (SLNs) and delivers them to their downstream LNs. In the protocol, we show that metastases in the proper axillary LN (PALN) are induced by the injection of luciferase-expressing tumor cells into the subiliac LN (SiLN) of MXH10/Mo-lpr/lpr (MXH10/Mo/lpr) mice. 5-FU is injected using the LDDS into the accessory axillary LN (AALN) to treat tumor cells in the PALN after the tumor cell growth is confirmed in the PALN. The tumor growth and therapeutic effects are evaluated by IVIS. This method can be used to evaluate tumor growth and efficacy of anticancer drugs/particles, radiotherapy, surgery, and/or a combination of these methods in various experimental procedures in the oncology field.

Abstract LBA048: Lymph node metastasis targeted intranodal delivery of docetaxel improves treatment outcome

Abstract Despite recent advances in diagnostic modalities and cancer treatment guidelines, systemic chemotherapy for the lymph node (LN) metastasis has poor treatment efficacy using conventional drugs. Therefore, a novel method, lymphatic drug delivery system (LDDS), has been proposed in a pre-clinical experiment that directly injects the anticancer drug into sentinel LNs to deliver into their downstream LNs, aiming to inhibit tumor cell growth in those LNs. The injection duration of chemotherapy drugs using the LDDS will be less than the intratumoral or intralesion methods used in the clinic, and systemic toxicity will be less than tumor-targeted therapy or systemic chemotherapy. Our previous results indicated that the treatment efficacy increased in LDDS treated groups than systemic delivery. Here, we show the improvement of treatment efficacy by increasing osmotic pressure and viscosity of the drug solvent delivered by LDDS using the metastatic LN mouse model. First, a metastatic LN mouse model was created by inoculating luciferase-expressing tumor cells into subiliac LN (SiLN) of MXH10/Mo/lpr mouse to induce metastasis into the proper axillary LN (PALN). Next, 10 mg/kg docetaxel (DTX) with increased osmotic pressure and viscosity were injected into tumor-inoculated SiLN with an injection rate of 2400 μL/min on day 7 post-inoculation to inhibit tumor cell growth in the inoculation and metastatic site. Luciferase activity decreases in PALNs and SiLNs were observed in groups between 1140 kPa, 1960 kPa, and 2780 kPa (4, 11, and 38 mPa·s) groups without inducing any side effects. By histology, no tumor cells were confirmed in those LNs of the 1960 kPa group. Then, DTX at 1960 kPa, 11 mPa·s was injected into tumor-inoculated SiLN on day 21 post-inoculation with different injection speeds and volumes. We found that DTX at 1960 kPa, 11 mPa·s injected at the injection rate of 200 μL/min with 400 μL volume, can inhibit tumor cell growth in the PALN and delay tumor growth SiLN. Furthermore, the antitumor effects depended on the tumor progression stage in the LNs, and the injection rates and volumes of administered drugs. We anticipate these optimal ranges as a starting point for developing more effective drug regimens to treat metastatic LNs with the LDDS. Moreover, administrable drugs in this novel method of ultrasound-guided LDDS or LDDS at intra-operative or image-guided surgery are not limited only to anticancer agents. Citation Format: Ariunbuyan Sukhbaatar, Shouta Sora, Mori Shiro, Tetsuya Kodama. Lymph node metastasis targeted intranodal delivery of docetaxel improves treatment outcome [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA048.

A Novel Brain-Permeant Chemotherapeutic Agent for the Treatment of Brain Metastasis in Triple-Negative Breast Cancer.

Development of metastases to central nervous system (CNS) is an increasing clinical issue following the diagnosis of advanced breast cancer. The propensity to metastasize to CNS varies by breast cancer subtype. Of the four breast cancer subtypes, triple-negative breast cancers (TNBC) have the highest rates of both parenchymal brain metastasis and leptomeningeal metastasis (LM). LM is rapidly fatal due to poor detection and limited therapeutic options. Therapy of TNBC brain metastasis and LM is challenged by multifocal brain metastasis and diffuse spread of LM, and must balance brain penetration, tumor cytotoxicity, and the avoidance of neurotoxicity. Thus, there is an urgent need for novel therapeutic options in TNBCs CNS metastasis. QBS10072S is a novel chemotherapeutic that leverages TNBC-specific defects in DNA repair and LAT1 (L-amino acid transporter type 1)-dependent transport into the brain. In our study, activity of QBS10072S was investigated in vitro with various cell lines including the human TNBC cell line MDA-MB-231 and its brain-tropic derivative MDA-MB-231-BR3. QBS10072S was preferentially toxic to TNBC cells. The efficacy of QBS10072S against brain metastasis and LM was tested using a model of brain metastasis based on the internal carotid injection of luciferase-expressing tumor cells into NuNu mice. The compound was well tolerated, delayed tumor growth and reduced leptomeningeal dissemination, resulting in significant extension of survival. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, QBS10072S is planned to be investigated in clinical trials as a therapeutic for TNBC LM. SIGNIFICANCE: TNBC brain metastasis often involves dissemination into leptomeninges. Treatment options for TNBC leptomeningeal metastasis are limited and are mostly palliative. Our study demonstrates significant efficacy of the brain-penetrating agent QBS10072S against TNBC brain metastasis and leptomeningeal spread.

Preclinical Applications of Multi-Platform Imaging in Animal Models of Cancer.

In animal models of cancer, oncologic imaging has evolved from a simple assessment of tumor location and size to sophisticated multimodality exploration of molecular, physiologic, genetic, immunologic, and biochemical events at microscopic to macroscopic levels, performed noninvasively and sometimes in real time. Here, we briefly review animal imaging technology and molecular imaging probes together with selected applications from recent literature. Fast and sensitive optical imaging is primarily used to track luciferase-expressing tumor cells, image molecular targets with fluorescence probes, and to report on metabolic and physiologic phenotypes using smart switchable luminescent probes. MicroPET/single-photon emission CT have proven to be two of the most translational modalities for molecular and metabolic imaging of cancers: immuno-PET is a promising and rapidly evolving area of imaging research. Sophisticated MRI techniques provide high-resolution images of small metastases, tumor inflammation, perfusion, oxygenation, and acidity. Disseminated tumors to the bone and lung are easily detected by microCT, while ultrasound provides real-time visualization of tumor vasculature and perfusion. Recently available photoacoustic imaging provides real-time evaluation of vascular patency, oxygenation, and nanoparticle distributions. New hybrid instruments, such as PET-MRI, promise more convenient combination of the capabilities of each modality, enabling enhanced research efficacy and throughput.

Analysis of tumor vascularization in a mouse model of metastatic lung cancer

Therapies targeting tumor vasculature would improve the treatment of lung metastasis, although the early changes in vascular structure are incompletely understood. Here, we show that obstructive metastatic foci in lung arterioles decrease the pulmonary vascular network. To generate a mouse model of lung metastasis activation, luciferase-expressing tumor cells were inoculated into the subiliac lymph node (SiLN) of an MXH10/Mo-lpr/lpr mouse, and metastatic tumor cells in the lungs were activated by SiLN resection. Activation of metastases was monitored by in vivo bioluminescence imaging. Pulmonary blood vessel characteristics were analyzed using ex vivo micro-computed tomography. The enhanced permeability and retention (EPR) effect in neovasculature after tumor cell activation was evaluated from the accumulation of intravenously injected indocyanine green (ICG) liposomes. Metastatic foci in lung arterioles were investigated histologically. Micro-computed tomography revealed decreases in pulmonary blood vessel length, volume and number of branching nodes during the early stage of metastasis caused by metastatic foci. ICG liposome accumulation by the EPR effect was not detected. Histology identified metastatic foci in lung arterioles. The lack of an EPR effect after the formation of metastatic foci in lung arterioles makes conventional systemic chemotherapy ineffective for lung metastasis. Thus, alternative therapeutic methods of drug delivery are needed.

Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Abstract 4066: AnnexinV: Multifunctional anticancer immunotherapeutic protein in tumor microenvironment

Abstract Introduction: After cancer treatment using chemotherapy and radiotherapy, tumor microenvironment continually occurs inflammation, which depend on damage-associated molecular pattern (DAMPs) released from apoptotic tumor cells. In this case, tumor microenvironment is regarded as inflammation zone and induce various immune cells infiltration, pro- and anti-inflammatory cytokines production and interaction between immune cells and cancer cells. These, a dynamic and complex microenvironment induce a favorable state for tumor growth. Thus, recent studies assert that it is important to regulate excessively increased inflammatory response in the tumor microenvironment accordingly. Annexin V is a Ca2+-dependent phospholipid binding pro[[Unsupported Character - Codename ­]]tein and known to bind to exposed phosphatidylserine on the apoptotic cells. Several groups and our study have recently demonstrated that annexin V has anti-inflammatory effects by inhibiting bone-marrow derived dendritic cell (BMDCs) activation and pro-inflammtory cytokine production. So, we suggest that Annexin V as a therapeutic approach for controlling inflammatory response in cancer patients having increased inflammation in tumor microenvironment. Methods: The size and purity of recombinant proteins were assessed using SDS-PAGE. IVIS imaging system used to characterize the accumulation of Annexin V containing protein into tumor loci in tumor-bearing mice. Therapeutic efficacy was confirmed tumor-specific CD8+ T cells in PBMCs and splenocytes by Flow cytometry analysis. pro-, anti- inflammtory cytokines and various immune cells infiltration in tumor microenvironment were examined by ELISA and FACS. Cytotoxicity by tumor-specific CD8+ T cells of Luciferase-expressing tumor cells was observed by representative luminescence imaging system. Results: The Targeting efficiency of Annexin V protein into tumor microenvironment was increased compared to control. Therapeutic efficacy was demostrated activation of tumor-specific CD8+ T cells (P < 0.001), inhibition of tumor growth (P < 0.05) and increase of survival (P < 0.05) in tumor bearing mouse compared to control. AnnexinV protein administration was decreased significantly TGF-beta 1 cytokine production (P < 0.001) and immunosuppressive cells infiltration (P < 0.001) in tumor microenviroment compared to non-treated group. Conclusion: We identified multiple role of Annexin V protein, which has capacity of apoptotic tumor cell target, as regulator of excessive inflammation in tumor microenvironment. This results could primarily pave the road for the development of anti-tumor immunotherapy. Citation Format: Jung Hwa Park, Young Seob Kim, Hyun Jin Park, Gun Young Jang, T.c. Wu, Yeong-Min Park, Tae Heung Kang. AnnexinV: Multifunctional anticancer immunotherapeutic protein in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4066.

Evaluation of the enhanced permeability and retention effect in the early stages of lymph node metastasis.

Most solid cancers spread to new sites via the lymphatics before hematogenous dissemination. However, only a small fraction of an intravenously administered anti‐cancer drug enters the lymphatic system to reach metastatic lymph nodes (LN). Here, we show that the enhanced permeability and retention (EPR) effect is not induced during the early stages of LN metastasis. Luciferase‐expressing tumor cells were injected into the subiliac LN of the MXH10/Mo‐lpr/lpr mouse to induce metastasis to the proper axillary LN (PALN). In vivo biofluorescence imaging was used to confirm metastasis induction and to quantify the EPR effect, measured as PALN accumulation of intravenously injected indocyanine green (ICG) liposomes. PALN blood vessel volume changes were measured by contrast‐enhanced high‐frequency ultrasound imaging. The volume and density of blood vessels in the PALN increased until day 29 after inoculation, whereas the LN volume remained constant. ICG retention was first detected on day 29 post‐inoculation. While CD31‐positive cells increased up to day 29 post‐inoculation, α‐smooth muscle actin‐positive cells were detected on day 29 post‐inoculation for the first time. These results suggest that the EPR effect was not induced in the early stages of LN metastasis; therefore, systemic chemotherapy would likely not be beneficial during the early stages of LN metastasis. The development of an alternative drug delivery system, independent of the EPR effect, is required for the treatment of LN metastasis.

Abstract 3195: Synergistic effects of combined treatment with hAT-MSC.sTRAIL and panobinostat in brainstem glioma

Abstract Human adipose-derived mesenchymal stem cells expressing secreted form of the tumor necrosis factor-related apoptosis-inducing ligand (hAT-MSC.sTRAIL) have demonstrated therapeutic activity against various tumors. However, sTRAIL resistance remains a challenge in developing anticancer strategies. To solve this problem, many studies have tried to combine drugs to produce synergism or sensitize resistant cancer cells. Histone deacetylase inhibitors (HDACi) have been known to induce expression of TRAIL death receptors 4 and 5 (DR4/DR5). Herein, we evaluated the use of combined therapy of hAT-MSC.sTRAIL with HDAC inhibitor, panobinostat, in enhancing sensitivity to hAT-MSC.sTRAIL mediated apoptosis against the brainstem glioma. A sTRAIL was introduced into the characterized hAT-MSCs using electroporation. To confirm appropriate treatment concentration of panobinostat to the glioblastoma cells, dose titration was tested using cell viability assay. The therapeutic effect of single or combination treatment against glioblastoma was evaluated using primary cultured glioblastoma cells and cell lines. Orthotopic xenograft brainstem glioma mouse model was established using engineered firefly luciferase expressing tumor cells for bioluminescence in vivo imaging. Panobinostat induced anti-proliferative effects in dose and time-dependent manner (IC50 range, 0.05-0.2 μM) and effectively enhanced the expression of TRAIL DR4 and DR5, but not decoy receptors. Combined hAT-MSC.sTRAIL and panobinostat significantly decreased the tumor cell growth compared to each alone. Intriguingly, the combination treatment not only induced apoptosis but also autophagy. Using a preclinical brainstem mouse model, we confirmed that the combination of hAT-MSC.sTRAIL and panobinostat was safe and induced regression of tumor volume. Furthermore, the combination therapy prolonged the survival of brainstem glioma-bearing mice. Our results suggested that combination therapy of panobinostat enhanced the anti-cancer effect of hAT-MSC.sTRAIL and represent potential therapeutic approach to the brainstem glioma. Citation Format: Seung Ah Choi, Chanhee Lee, Pil Ae Kwak, Kyu-Chang Wang, Ji Hoon Phi, Ji Yeoun Lee, Sangjoon Chong, Seung-Ki Kim. Synergistic effects of combined treatment with hAT-MSC.sTRAIL and panobinostat in brainstem glioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3195.

Abstract B60: Role of inflammatory monocyte mobilization in metastatic pancreatic cancer

Abstract Introduction: More than half of patients with Pancreatic Cancer (PC) present with metastasis. Our group and others have shown that PC induces cellular changes in the liver long before metastatic spread. These pre-metastatic changes include a significant recruitment of inflammatory monocytes (IM) by the chemokine CCL2 (produced by pre-metastatic liver) and its receptor CCR2 (expressed on IM). Based on our preliminary data, we hypothesized that IM mobilization to the liver is essential for growth of metastatic PC. We therefore optimized our model of metastatic PC to study the role of CCR2 inhibition on growth of metastatic PC. Methods: We implanted a spontaneously derived murine PC cell line (KCKO) orthotopically in the tail of the pancreas of C57BL/6 (WT) mice. We removed the primary tumor on day 10 and divided the spleen into two hemi-spleens. At this time, mice were injected with luciferase expressing tumor cells in the inferior pole of the spleen which was removed after injection and liver metastases were detected by bioluminescence (BLI). Results: Mice with established liver metastasis were randomized to treatment with CCR2 antagonist (CCR2i), FOLFIRINOX, CCR2i and FOLFIRINOX combination (CCR2i+FOLFIRINOX), or vehicle. Mice were treated for three weeks and were imaged biweekly in order to quantify tumor burden in the liver. Mice were sacrificed after 3 weeks and gene expression and flow cytometry studies were performed on peripheral blood, bone marrow, and liver. Liver metastases were detected by BLI in 70% of mice after 10 days and 100% of mice after 15 days following splenic injection with luciferase expressing tumor cells. Peripheral blood and liver inflammatory monocytes/macrophages were significantly increased in mice bearing liver metastasis. However, CCR2i efficiently blocked the recruitment of inflammatory monocyte/macrophage populations in the liver (p<0.01). Additionally mice treated with CCR2i alone and CCR2i and FOLFIRINOX combination exhibited a shift in the immune gene profile in metastatic liver from a pro-tumor (Type 2) to an anti-tumor (Type 1) immune response. Consequently, metastatic tumor burden was significantly decreased after treatment with CCR2i and FOLFIRINOX. Interestingly, the lowest tumor burden was found in livers of mice treated with combination of CCR2 inhibitor and FOLFIRINOX. This suggests that targeting CCR2 can decrease the growth of liver metastasis in PC. Conclusion: Our murine model of PC metastasis formation recapitulates the human form of the disease. We demonstrate that inflammatory monocyte recruitment to the liver is important in promoting the development of metastasis in liver. CCR2 blockade in combination with FOLFIRINOX decreases macrophages in the liver and impairs growth of liver metastasis. Citation Format: Roheena Z. Panni, Dominic E. Sanford, Brian A. Belt, Timothy M. Nywening, Peter Goedegebuure, David C. Linehan. Role of inflammatory monocyte mobilization in metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B60.

Temporal Dynamics of Tumor-Microenvironment Interaction and Treatment Responses Revealed through Time-Lapse Compartment-Specific Bioluminescence Imaging: Translational implications

Most conventional methods to sensitively quantify tumor cell proliferation and viability in vitro involve processing of cells in ways that preclude continuation of the respective experiment or prevent the longitudinal collection of data. This common technical feature of conventional assays limits their ability to provide detailed insight into the kinetics of tumor cell responses to treatment(s). Additionally, these limitations hinder the use of these assays to monitor how the kinetics of treatment response can be altered by nonmalignant “accessory” cells of the tumor microenvironment (e.g. bone marrow stromal cells [BMSCs] for hematologic malignancies or bone metastases of solid tumors). To address these obstacles, we modified our previously developed tumor cell compartment-specific bioluminescence imaging (CS-BLI) platform (McMillin et al. Nat Med. 2010), to enable longitudinal assessment of tumor cell response to diverse experimental conditions; we cultured luciferase-expressing tumor cells, with or without stromal cells, in the presence of bioluminescent substrates, using optimized conditions which provide detectable bioluminescent signal even after several days of culture, while having no adverse effect on the viability of tumor or non-malignant cells in this system. This modified approach (time-lapse CSBLI, [TL-CSBLI]) preserved the linear correlation of bioluminescent signal with tumor cell viability. Furthermore, results obtained at the end of the experiment and during interim time-points are consistent with those generated using either non-time-lapse applications of CS-BLI or conventional techniques. We applied TL-CSBLI to delineate, in high-throughput manner, the temporal dynamics of the responses of tumor cells (e.g. multiple myeloma (MM) and other hematologic malignancies) to diverse treatments (e.g. conventional chemotherapeutics, glucocorticoids; proteasome inhibitors (PIs, bortezomib or carfilzomib), and kinase inhibitors). Using the time-lapse capabilities of this assay, we evaluated tumor cell responses in the presence vs. absence of stromal cells. We observed that the kinetics of tumor cell response to diverse therapeutic classes are heterogeneous, even within the same tumor type: for instance, tumor cells with pronounced responses at the end of drug incubation (e.g. 24, 48, 72, hrs after initiation of treatment with PIs, DNA-damaging chemotherapeutics, or dexamethasone respectively), can have different magnitude of responses at intermediate time points. This suggests that TL-CSBLI data can further stratify treatment-responsive tumor cells into those with early vs. late kinetics of response. We also observed that the kinetics of the proliferative / anti-apoptotic effect conferred by stromal cells on tumor cells are highly variable between different cell lines, even within the same tumor type. For instance, the time between initiation of coculture and maximum stimulation of tumor cell viability by stromal cells was variable between cell lines and did not correlate with the magnitude of stimulation by stromal cells. Importantly, TL-CSBLI identified that the response of diverse types of tumor cells to treatments can be delayed in the presence of stromal cells, compared to conventional tumor cell monocultures: this initial delay in treatment response of tumor cells in stromal co-cultures may be observed even in cases where similar cytoreductive responses are eventually observed at later time-points in both the presence and absence of stromal cells. This observation suggests that a more expansive definition of stroma-induced resistance to a given treatment may be warranted, to specifically incorporate the ability of stromal cells to delay the tumor cell response to such treatment. In summary, TL-CSBLI enables detailed characterization of the kinetics of tumor cell responses to diverse experimental conditions. Its use can provide insight into the underappreciated impact that cell-autonomous variations or stroma-induced changes in the kinetics of tumor cell response to a given anti-tumor therapy can have on determining its efficacy. This is particularly consequential for agents (e.g. PIs) which have clinical pharmacokinetic profiles associated with transient peak exposure. DisclosuresMcMillin:Axios Biosciences: Equity Ownership; DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Negri:DFCI: patent submission on stromal co-culture technologies Patents & Royalties. Mitsiades:Johnson & Johnson: Research Funding; Amgen: Research Funding; Celgene: Consultancy, Honoraria; Millennium Pharmaceuticals: Consultancy, Honoraria; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

AbstractAbstract 451Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT.We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells.In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ε, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Integrated Imaging Approach to Tumor Model Mice Using Bioluminescence Imaging and Magnetic Resonance Imaging

We developed an integrated imaging method using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) and applied it to mouse tumor monitoring. BLIs and MRIs were successively acquired from mice fixed on a transparent flat plate and were registered using three small markers. After spatial coregistration, the two-dimensional BLI was fused with all MRI slices. The accuracy of image registration was evaluated using fluorescence imaging and MRI of many markers attached to the mice. Mice were inoculated with luciferase-expressing tumor cells subcutaneously or directly into the liver, and disease progression was evaluated repeatedly by integrated BLI-MRI. Image registration was achieved with acceptable accuracy, although the registration error increased with increasing distance from the center of the imaging field and was larger in imaging the convex dorsal side of the mouse than in imaging the flat ventral side. Integrated BLI-MRI allowed longitudinal assessment of disease progression and appeared to facilitate detailed interpretation of the imaging findings. It aided the anatomic localization of lesions demonstrated by BLI and recognition of MRI abnormalities corresponding to BLI foci. Integrated BLI-MRI is feasible and appears to be a practical method for comprehensive, longitudinal assessment of disease model mice.

Oncogenic K-Ras Turns Death Receptors Into Metastasis-Promoting Receptors in Human and Mouse Colorectal Cancer Cells

Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein- or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis. Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formation.

Neural Progenitor Cell-mediated Delivery of Interferon Beta Improves Neuroblastoma Response to Cyclophosphamide

We have shown that continuous systemic delivery of interferon beta (IFN-beta) remodels dysfunctional tumor vasculature, thereby improving tumor perfusion and enhancing delivery and efficacy of chemotherapeutic drugs. We hypothesized that because of their inherent tumor tropism, neural progenitor cells (NPCs) engineered to express IFN-beta could also effect maturation of tumor vasculature without generating high systemic levels of IFN-beta. Mice with luciferase-expressing disseminated human neuroblastoma were divided into four groups of equal tumor burden by bioluminescence imaging: (1) untreated controls; (2) NPC-IFN-beta only; (3) cyclophosphamide (CTX) only; and (4) NPC-IFN-beta in combination with CTX. Two million NPC-IFN-beta cells were administered twice, 7 days apart, starting 21 days after tail vein administration of tumor cells. CTX was administered every 6 days for three doses. Mice were killed at 6 weeks, livers and kidneys weighed, and tumor removed for immunohistochemical staining for endothelial cells (CD34), pericytes (alpha-SMA), apoptosis (TUNEL [terminal deoxynucleotidyl transferase dUTP nick-end labeling]), and diI-labeled NPCs. Fluorescent-labeled NPCs confirmed localization of these cells to tumors. The alpha-SMA/CD34 ratio, a marker for vascular maturation, greatly increased in NPC-IFN-beta-treated tumors compared with controls. Bioluminescent signal from luciferase-expressing tumor cells, reflecting tumor burden, was lower with combination therapy than control or either monotherapy, and combination therapy resulted in far less tumor burden by weight in the kidneys and liver. Targeted delivery of IFN-beta with NPCs produced low circulating levels of IFN-beta, yet the maturing effect on the tumor vasculature and the enhanced efficacy of adjuvant therapy was maintained. Thus, combination therapy of NPC-IFN-beta with CTX warrants further investigation for the treatment of high-risk neuroblastoma patients.

Bevacizumab suppresses neuroblastoma progression in the setting of minimal disease

We hypothesized that vascular endothelial growth factor (VEGF) contributes to autocrine stimulation of neuroblastoma and that inhibition of its signaling pathway contributes to the anticancer activity of bevacizumab, an anti-VEGF monoclonal antibody. For in vitro studies, 2 neuroblastoma cell lines, CHLA-255 and NB1691, were treated with VEGF+/-bevacizumab. For in vivo studies, disseminated neuroblastoma was established by intravenous administration of luciferase-expressing tumor cells in SCID mice prior to bevacizumab treatment. Exogenous VEGF increased cell counts after 48 h (NB1691: 58,878 +/- 8279 vs 137,500 +/- 13,108 cells, P < .001; CHLA: 1.56 x 10(6) +/- 866 vs 1.81 x 10(6) +/- 2550 cells, P <.001); the addition of bevacizumab abrogated this stimulation. In vivo, mice with disseminated disease treated twice weekly with intraperitoneal bevacizumab had a decreased tumor burden at day 14 and prolonged survival (NB1691: 50 +/- 2 vs 43 +/- 2 days, P < .001; CHLA: 53 +/- 3 vs 42 +/- 1 days, P = .006). Interestingly, VEGF and basic fibroblast growth factor expression was increased in treated NB1691 tumors, which likely occurred in response to VEGF signaling inhibition. Our results suggest that VEGF has a role in neuroblastoma autocrine signaling. Maintenance therapy with bevacizumab may be useful for disease suppression after maximal cytoreductive therapy; however, upregulation of proangiogenic factors may provide resistance to this approach, which suggests that maximal antitumor efficacy may require combination therapy.

Use of bioluminescence imaging to detect enhanced hepatic and systemic tumor growth following partial hepatectomy in mice

The impact of partial hepatectomy on intra-hepatic and distant tumor growth is a matter of controversy. Utilizing a highly sensitive tumor imaging strategy, we sought to demonstrate whether this growth-acceleration occurs, and to develop an animal model with which to investigate potential therapeutic strategies. Mice bearing constitutively-active luciferase-expressing tumor cells were subjected to either 70% partial hepatectomy (PH; n=10) or a sham operation (n=11). Mice were sacrificed 14 days later and remnant livers (or anatomic equivalents in the control group) and lungs harvested for bioluminescence detection. Remnant liver weights were significantly increased in PH compared to equivalent lobes in sham-operated animals (t-test; p=0.005). Tumor burden as measured by bioluminescence was significantly higher in both liver and lung specimens in the PH group (Wilcoxon's Rank Sum test; p=0.01 and 0.004, respectively). Following PH, enhanced metastatic growth was depicted regionally and systemically with bioluminescence imaging providing an objective measure of tumor burden. This preclinical model can help to identify adjuvant therapies that can influence both tumor growth and liver regeneration.

Optical Imaging of Tumor Cells in Hollow Fibers: Evaluation of the Antitumor Activities of Anticancer Drugs, Target Validation

The in vivo hollow fiber assay, in which semipermeable hollow fibers filled with tumor cells, are implanted into animals, was originally developed to screen for anticancer compounds before assessment in more complex tumor models. To enhance screening, evaluation of anticancer drugs, we have applied optical imaging technology to this assay. To demonstrate that tumor cells inside hollow fibers can communicate with the host mice, we have used fluorescence imaging in vivo, CD31 immunostaining ex vivo to show that angiogenesis occurs around cell-filled hollow fibers by 2 weeks after subcutaneous implantation. Bioluminescence imaging has been used to follow the number of luciferase-expressing tumor cells within implanted hollow fibers; proliferation of those cells was found to be significantly inhibited by docetaxel, irinotecan. We also used bioluminescence imaging of hollow fibers to monitor the nuclear factor κB (NFκB) pathway in vivo; NFκB activation by lipopolysaccharide, tumor necrosis factor-α was evaluated in tumor cell lines genetically engineered to express luciferase controlled by an NFκB-responsive element. These results demonstrate that optical imaging of hollow fibers containing reporter tumor cells can be used for the rapid, accurate evaluation of antitumor activities of anticancer drugs, for measurement of molecular pathways.