Non-Invasive Direct Visualization of Luciferase-Luciferin Emitted Light Producing True Images from an Orthotopic Lung Cancer Xenograft Model in Nude Mice Using an Ultra-sensitive Low-light Camera and Optics.

BACKGROUND/AIM: In vivo imaging with luciferase-luciferin has been limited by the inability to visualize the low emitted light, with the signal quantified only by photon counting using a cumbersome highly-cooled CCD camera in a dark room. In the present study, we demonstrate direct visualization of the luciferase-luciferin signal from an orthotopic lung cancer in a nude-mouse xenograft model with a sensitive low-light camera and optics. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells expressing luciferase (LL/2-Luc2) were injected transcutaneously into the lung of a nude mouse. One week later after cell injection, luciferase imaging for emission at 560 nm was performed using the UVP Biospectrum Advanced system after i.v. injection of D-luciferin potassium salt. The intensity of the visualized light was measured and quantified with the instrument. RESULTS: A week following the implantation of LL/2-Luc2 cells in nude mice, the luciferase-luciferin signal from LL/2-Luc2 tumors in the lung was sufficiently visible through the skin to produce true images. At fifteen minutes, the intensity peaked and then progressively dropped due to clearance of luciferin from the tumor. CONCLUSION: Using the UVP Biospectrum Advanced system we demonstrated non-invasive visualization of true images from luciferase-luciferin signals from an orthotopic lung-cancer mouse model. The luciferase-luciferin emitted light was directly visible through the skin which is a major improvement over previous photon counting to detect the luciferase-luciferin signal.

A Novel Luciferase-Based Reporter Gene Technology for Simultaneous Optical and Radionuclide Imaging of Cells.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

Rapid function analysis of OsiWAK1 using a Dual-Luciferase assay in rice.

In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study.

A Novel Method for the Early Detection of Single Circulating, Metastatic and Self-Seeding Cancer Cells in Orthotopic Breast Cancer Mouse Models.

BACKGROUND: Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. METHODS: Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. RESULTS: In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. CONCLUSIONS: These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems.

The ability of proteins to sense and transmit mechanical forces underlies many biological processes, but characterizing these forces in biological systems remains a challenge. Existing genetically encoded force sensors typically rely on fluorescence or bioluminescence resonance energy transfer (FRET or BRET) to visualize tension. However, these force sensing modules are relatively large, and interpreting measurements requires specialized image analysis and careful control experiments. Here, we report a compact molecular tension sensor that generates a bioluminescent signal in response to tension. This sensor (termed PILATeS) makes use of the split NanoLuc luciferase and consists of the H. sapiens titin I10 domain with the insertion of a 10-15 amino acid tag derived from the C-terminal ß-strand of NanoLuc. Mechanical load across PILATeS mediates exposure of this tag to recruit the complementary split NanoLuc fragment, resulting in force-dependent bioluminescence. We demonstrate the ability of PILATeS to report biologically meaningful forces by visualizing forces at the interface between integrins and extracellular matrix substrates. We further use PILATeS as a genetically encoded sensor of tension experienced by the mechanosensing protein vinculin. We anticipate that PILATeS will provide an accessible means of visualizing molecular-scale forces in biological systems.

In vitro screening of chemically synthesized dipeptide-antisense oligonucleotide conjugates to identify ligand molecules enhancing their activity.

Oligonucleotide therapeutics, particularly antisense oligonucleotides (ASOs), have emerged as promising candidates in drug discovery. However, their effective delivery to the target tissues and cells remains a challenge, necessitating the development of suitable drug delivery technologies for ASOs to enable their practical application. In this study, we synthesized a library of chemically modified dipeptide-ASO conjugates using a recent synthetic method based on the Ugi reaction. We then conducted in vitro screening of this library using luciferase-expressing cell lines to identify ligands capable of enhancing ASO activity. Our findings suggest that N-(4-nitrophenoxycarbonyl)glycine may interact with the thiophosphate moiety of the phosphorothioate-modification in ASO. Through our screening efforts, we identified two ligands that modestly reduced luciferase luminescence in a cell type-selective manner. Furthermore, quantification of luciferase mRNA levels revealed that one of these promising dipeptide-ASO conjugates markedly suppressed luciferase RNA levels through its antisense effect in prostate-derived DU-145 cells compared to the ASOs without ligand modification.

Emerging Synthetic Bioluminescent Reactions for Non-Invasive Imaging of Freely Moving Animals.

Bioluminescence imaging (BLI) is an indispensable technique for visualizing the dynamics of diverse biological processes in mammalian animal models, including cancer, viral infections, and immune responses. However, a critical scientific challenge remains: non-invasively visualizing homeostatic and disease mechanisms in freely moving animals to understand the molecular basis of exercises, social behavior, and other phenomena. Classical BLI relies on prolonged camera exposure to accumulate the limited number of photons that traveled from deep tissues in anesthetized or constrained animals. Recent advancements in synthetic bioluminescence reactions, utilizing artificial luciferin-luciferase pairs, have considerably increased the number of detectable photons from deep tissues, facilitating high-speed BLI to capture moving objects. In this review, I provide an overview of emerging synthetic bioluminescence reactions that enable the non-invasive imaging of freely moving animals. This approach holds the potential to uncover unique physiological processes that are inaccessible with current methodologies.

A suicidal and extensively disordered luciferase with a bright luminescence.

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate-binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

A pH-sensitive closed-loop nanomachine to control hyperexcitability at the single neuron level.

Epilepsy affects 1% of the general population and 30% of patients are resistant to antiepileptic drugs. Although optogenetics is an efficient antiepileptic strategy, the difficulty of illuminating deep brain areas poses translational challenges. Thus, the search of alternative light sources is strongly needed. Here, we develop pH-sensitive inhibitory luminopsin (pHIL), a closed-loop chemo-optogenetic nanomachine composed of a luciferase-based light generator, a fluorescent sensor of intracellular pH (E2GFP), and an optogenetic actuator (halorhodopsin) for silencing neuronal activity. Stimulated by coelenterazine, pHIL experiences bioluminescence resonance energy transfer between luciferase and E2GFP which, under conditions of acidic pH, activates halorhodopsin. In primary neurons, pHIL senses the intracellular pH drop associated with hyperactivity and optogenetically aborts paroxysmal activity elicited by the administration of convulsants. The expression of pHIL in hippocampal pyramidal neurons is effective in decreasing duration and increasing latency of pilocarpine-induced tonic-clonic seizures upon in vivo coelenterazine administration, without affecting higher brain functions. The same treatment is effective in markedly decreasing seizure manifestations in a murine model of genetic epilepsy. The results indicate that pHIL represents a potentially promising closed-loop chemo-optogenetic strategy to treat drug-refractory epilepsy.

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-based Reporter Assays.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli. Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence. These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Establishment of the Foot-and-Mouth Disease Virus Type Asia1 Expressing the HiBiT Protein: A Useful Tool for a NanoBiT Split Luciferase Assay.

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that affects cloven-hoofed animals and causes severe economic losses in the livestock industry. Given that this high-risk pathogen has to be handled in a biosafety level (BSL)-3 facility for safety reasons and the limited availability of BSL-3 laboratories, experiments on FMDV call for more attention. Therefore, we aimed to develop an FMDV experimental model that can be handled in BSL-2 laboratories. The NanoBiT luciferase (Nano-luc) assay is a well-known assay for studying protein-protein interactions. To apply the NanoBiT split luciferase assay to the diagnosis and evaluation of FMD, we developed an inactivated HiBiT-tagged Asia1 Shamir FMDV (AS-HiBiT), a recombinant Asia1 shamir FMDV with HiBiT attached to the VP1 region of Asia1 shamir FMDV. In addition, we established LgBiT-expressing LF-BK cell lines, termed LgBit-LF-BK cells. It was confirmed that inactivated AS-HiBiT infected LgBiT-LF-BK cells and produced a luminescence signal by binding to the intracellular LgBiT of LgBiT-LF-BK cells. In addition, the luminescence signal became stronger as the number of LgBiT-LF-BK cells increased or the concentration of inactivated AS-HiBiT increased. Moreover, we confirmed that inactivated AS-HiBiT can detect seroconversion in sera positive for FMDV-neutralizing antibodies. This NanoBiT split luciferase assay system can be used for the diagnosis and evaluation of FMD and expanded to FMD-like virus models to facilitate the evaluation of FMDV vaccines and antibodies.

Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts.

Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 µg/mL luciferase in batch reactions and 60 ± 4 µg/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

Head-to-head Comparison of Green Fluorescent Protein (GFP) Imaging With Luciferase-luciferin Imaging In Vivo Using Single-nanometer Laser-excitation Tuning and an Ultra-low-light-detection Camera and Optics Demonstrates the Superiority of GFP.

BACKGROUND/AIM: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. MATERIALS AND METHODS: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. RESULTS: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. CONCLUSION: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.

Development of a split-luciferase assay to establish optimal protein secretion conditions for protein production by Bacillus subtilis.

Bacillus subtilis is a Gram-positive bacterium that is frequently used in the bioindustry for the production of various proteins, because of its superior protein secretion capacities. To determine optimal conditions for protein secretion by B. subtilis, a quick and sensitive method for measuring protein secretion is crucial. A fast and universal assay is most useful for detecting diverse proteins in a high-throughput manner. In this study, we introduce a split-luciferase-based method for measuring protein secretion by B. subtilis. The NanoBiT system was used to monitor secretion of four different proteins: xylanase A, amylase M, protein glutaminase A, and GFP nanobody. Our findings underscore the split-luciferase system as a quick, sensitive, and user-friendly method.

Demystifying In Vivo Bioluminescence Imaging of a Chagas Disease Mouse Model for Drug Efficacy Studies.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

Low-cost and sustainable smartphone-based tissue-on-chip device for bioluminescence biosensing.

Several organ-on-chip and cell-on-chip devices have been reported, however, their main drawback is that they are not interoperable (i.e., they have been fabricated with customized equipment, thus cannot be applied in other facilities, unless having the same setup), and require cell-culture facilities and benchtop instrumentation. As a consequence, results obtained with such devices do not generally comply with the principles of findability, accessibility, interoperability, and reusability (FAIR). To overcome such limitation, leveraging cost-effective 3D printing we developed a bioluminescent tissue on-a-chip device that can be easily implemented in any laboratory. The device enables continuous monitoring of cell co-cultures expressing different bioluminescent reporter proteins and, thanks to the implementation of new highly bioluminescent luciferases having high pH and thermal stability, can be monitored via smartphone camera. Another relevant feature is the possibility to insert the chip into a commercial 24-well plate for use with standard benchtop instrumentation. The suitability of this device for 3D cell-based biosensing for monitoring activation of target molecular pathways, i.e., the inflammatory pathway via nuclear factor kappa-B (NF-κB) activation, and general cytotoxicity is here reported showing similar analytical performance when compared to conventional 3D cell-based assays performed in 24-well plates.

Maintain the light, long-term seasonal monitoring of luminous capabilities in the brittle star Amphiura filiformis.

The European brittle star Amphiura filiformis emits blue light, via a Renilla-like luciferase, which depends on the dietary acquisition of coelenterazine. Questions remain regarding luciferin availability across seasons and the persistence of luminous capabilities after a single boost of coelenterazine. To date, no study has explored the seasonal, long-term monitoring of these luminous capabilities or the tracking of luciferase expression in photogenic tissues. Through multidisciplinary analysis, we demonstrate that luminous capabilities evolve according to the exogenous acquisition of coelenterazine throughout adult life. Moreover, no coelenterazine storage forms are detected within the arms tissues. Luciferase expression persists throughout the seasons, and coelenterazine's presence in the brittle star diet is the only limiting factor for the bioluminescent reaction. No seasonal variation is observed, involving a continuous presence of prey containing coelenterazine. The ultrastructure description provides a morphological context to investigate the green autofluorescence signal attributed to coelenterazine during luciferin acquisition. Finally, histological analyses support the hypothesis of a pigmented sheath leading light to the tip of the spine. These insights improve our understanding of the bioluminescence phenomenon in this burrowing brittle star.

KPC-luciferase-expressing cells elicit an anti-tumor immune response in a mouse model of pancreatic cancer.

Mouse models for the study of pancreatic ductal adenocarcinoma (PDAC) are well-established and representative of many key features observed in human PDAC. To monitor tumor growth, cancer cells that are implanted in mice are often transfected with reporter genes, such as firefly luciferase (Luc), enabling in vivo optical imaging over time. Since Luc can induce an immune response, we aimed to evaluate whether the expression of Luc could affect the growth of KPC tumors in mice by inducing immunogenicity. Although both cell lines, KPC and Luc transduced KPC (KPC-Luc), had the same proliferation rate, KPC-Luc tumors had significantly smaller sizes or were absent 13 days after orthotopic cell implantation, compared to KPC tumors. This coincided with the loss of bioluminescence signal over the tumor region. Immunophenotyping of blood and spleen from KPC-Luc tumor-bearing mice showed a decreased number of macrophages and CD4+ T cells, and an increased accumulation of natural killer (NK) cells in comparison to KPC tumor mice. Higher infiltration of CD8+ T cells was found in KPC-Luc tumors than in their controls. Moreover, the immune response against Luc peptide was stronger in splenocytes from mice implanted with KPC-Luc cells compared to those isolated from KPC wild-type mice, indicating increased immunogenicity elicited by the presence of Luc in the PDAC tumor cells. These results must be considered when evaluating the efficacy of anti-cancer therapies including immunotherapies in immunocompetent PDAC or other cancer mouse models that use Luc as a reporter for bioluminescence imaging.