Mammalian Cell Culture Research Articles

Evolution Driven Microscale Combinatorial Chemistry in Intracellular Mimicking Droplets to Engineer Thermostable RNA for Cellular Imaging.

Fluorescent light-up aptamer/fluorogen pairs are powerful tools for tracking RNA in the cell, however limitations in thermostability and fluorescence intensity exist. Current in vitro selection techniques struggle to mimic complex intracellular environments, limiting in vivo biomolecule functionality. Taking inspiration from microenvironment-dependent RNA folding observed in cells and organelle-mimicking droplets, an efficient system is created that uses microscale heated water droplets to simulate intracellular conditions, effectively replicating the intracellular RNA folding landscape. This system is integrated with microfluidic droplet sorting to evolve RNA aptamers. Through this approach, an RNA aptamer is engineered with improved fluorescence activity by exploring the chemical fitness landscape under biomimetic conditions. The enhanced RNA aptamer named eBroccoli has increased fluorescence intensity and thermal stability, both in vitro and in vivo in bacterial and mammalian cells. In mammalian cell culture conditions, a fluorescence improvement of 3.9-times is observed and biological thermal stability up to 45°C is observed in bacterial systems. eBroccoli enable real-time visualization of nanoscale stress granule formation in mammalian cells during heat shock at 42°C. By introducing the concept of "biomimetic equivalence" based on RNA folding, the platform offers a simple yet effective strategy to mimic intracellular complexity in evolution-based engineering.

Bicarbonate Within: A Hidden Modulator of Antibiotic Susceptibility.

Since its standardization, clinical antimicrobial susceptibility testing (AST) has relied upon a standard medium, Mueller-Hinton Broth/Agar (MHB/A), to determine antibiotic resistance. However, this microbiologic medium bears little resemblance to the host milieu, calling into question the physiological relevance of resistance phenotypes it reveals. Recent studies investigating antimicrobial susceptibility in mammalian cell culture media, a more host-mimicking environment, demonstrate that exposure to host factors significantly alters susceptibility profiles. One such factor is bicarbonate, an abundant ion in the mammalian bloodstream/tissues. Importantly, bicarbonate sensitizes methicillin-resistant Staphylococcus aureus (MRSA) to early-generation β-lactams used for the treatment of methicillin-susceptible S. aureus (MSSA). This "NaHCO3-responsive" phenotype is widespread among US MRSA USA300/CC8 bloodstream and skin and soft tissue infection isolates. Translationally, β-lactam therapy has proven effective against NaHCO3-responsive MRSA in both ex vivo simulated endocarditis vegetation (SEV) and in vivo rabbit infective endocarditis (IE) models. Mechanistically, bicarbonate appears to influence mecA expression and PBP2a production/localization, as well as key elements for PBP2a functionality, including the PBP2a chaperone PrsA, components of functional membrane microdomains (FMMs), and wall teichoic acid (WTA) synthesis. The NaHCO3-responsive phenotype highlights the critical role of host factors in shaping antibiotic susceptibility, emphasizing the need to incorporate more physiological conditions into AST protocols.

Scale-up of CHO cell cultures: from 96-well-microtiter plates to stirred tank reactors across three orders of magnitude.

For process development in mammalian cell cultivations, scale-up approaches are essential. A lot of studies concern the scale transfer between different-sized stirred tank reactors. However, process development usually starts in even smaller cultivation vessels like microtiter plates or shake flasks. A scale-up from those small shaken devices to a stirred tank reactor is barely stated in literature for mammalian cells. Thus, this study aims to address data-driven scale-up for CHODP12 cells. The oxygen transfer rate is used as a database. The cultivation conditions in microtiter plates and shake flasks are comparable when choosing the maximum oxygen transfer capacity as a scale-up parameter. The minimum cultivation volume was reduced to 400µL in round and square 96-deep-well microtiter plates. Using a scale-up based on the maximum oxygen transfer capacity to a stirred tank reactor led to conditions with excessive hydromechanical stress. However, cultivation conditions could be reproduced in a stirred tank reactor by utilizing the volumetric power input as a scale-up parameter. Key metabolites behaved the same in all three scales and the final antibody titer was equal. This study presents a successful replication of cultivation results for mammalian cells in microtiter plates, shake flasks and stirred tank reactors. The working volumes ranged from 0.4 to 50 and 600mL. It offers the opportunity to adapt the method to other, more sensitive mammalian cells and to perform cost- and time-effective experiments in high-throughput.

Evaluating the impact of media and feed combinations on CHO cell culture performance and monoclonal antibody (trastuzumab) production

The choice of media and feeds significantly influences the performance of Chinese Hamster Ovary (CHO) mammalian cell cultures in producing desired biologics like monoclonal antibodies (mAb). Sub-optimal nutrient feed/media composition can severely impact cell proliferation and the quality of the final mAb product. For instance, proper protein glycosylation, crucial for mAb stability, safety, and efficacy, heavily relies on cell culture conditions. Currently, starter CHO culture media and daily supplemental feeds used in industrial manufacturing consist of proprietary composition of nutrients critical for mAb production. Standardized optimal media/feed combinations necessary for different cell lines are often lacking, necessitating individualized optimization for each cell line and mAb product. Here, we focused on a CHO-K1 cell line engineered to produce a Trastuzumab biosimilar and evaluated the effects of fourteen commercially relevant basal media and seven feeds on cell culture parameters such as viable cell density, viability, nutrient consumption, metabolite production, mAb titer, and mAb N-glycosylation. Our findings demonstrate clearly that the compositions of the basal medium and feed play a pivotal role in enhancing cell growth and mAb production. This work offers valuable insights into strategies for optimizing feed/media composition for glycosylated monoclonal antibody production using CHO cells.

CellREADR: An ADAR-based RNA sensor-actuator device.

RNAs are central mediators of genetic information flow and gene regulation that underlie diverse cell types and cell states across species. Thus, methods that can sense and respond to RNA profiles in living cells will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' protein payload coding region. Payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A-to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus represents a new generation of programmable RNA device for monitoring and manipulating animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed procedure for implementing CellREADR experiments in cell culture systems and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo protocol focuses on AAV-based delivery of CellREADR through expression vectors using brain tissue as an example. We describe current best practices and various experimental controls.

Virus Filtration Development for Adeno-Associated Virus-Based Gene Therapy Products.

Adeno-associated virus (AAV) vectors have become a leading platform for gene delivery. A major portion of gene therapy currently in clinical trials are AAV-based for a wide range of diseases. A commonly used method for AAV production is by mammalian or insect cell culture, with or without added viruses to introduce needed genetic elements for AAV production. There are potential risks of virus contamination from the production cell line, process residuals, or adventitious contamination in the production of biotherapeutics, including AAV-based gene therapy products; therefore, it is imperative to demonstrate that the drug substance manufacturing process has sufficient capability to clear process-related or adventitious viruses. In the AAV-based gene therapy manufacturing process, cell lysis, affinity chromatography, and ion exchange chromatography steps are often effective to inactivate or remove viruses. To increase the viral clearance robustness, virus filtration (VF) is increasingly recommended by regulatory agencies for gene therapy products as a dedicated viral clearance step in the downstream purification process. In the current study, two commercially available virus filters were evaluated in the context of AAV manufacturing. The filter throughput and process yield were assessed under different operational modes. Virus clearance performance was evaluated by spiking in Adenovirus type 5 (Adv-5) and Simian virus 40 (SV-40). The viral filters assessed in this study demonstrated manufacturable throughputs, acceptable process yields, and robust virus clearance capabilities for viruses greater than 40nm.

Cell culture research in aging and Alzheimer's disease: The strategic use/reuse of untreated controls and savings people's tax dollars

Cell culture is an essential tool in both fundamental and translational research, particularly for understanding complex diseases like Alzheimer's disease (AD). The use of cell lines provides the advantage of genetic homogeneity, ensuring reproducible and consistent results. This article explores the application of mammalian cell cultures to model AD, focusing on the transfection of cells with key genes associated with the disease to replicate the cellular environment of AD. It explains various transfection methods and challenges related to the process. These models offer a robust platform for investigating cellular biology, molecular pathways, physiological processes, and drug discovery efforts. A range of assays, including RT-PCR, western blotting, ELISA, mitochondrial respiration, and reactive oxygen species analysis, are employed to assess the impact of genetic modifications on cellular functions and to screen potential AD therapies. Researchers often design experiments with multiple variables such as genetic modifications, chemical treatments, or time points, paired with positive and negative controls. By using a consistent control group across all conditions and under identical experimental conditions, researchers can minimize variability and enhance data reproducibility. This approach is particularly valuable in AD research, where small experimental differences can significantly influence outcomes. Using a shared control group ensures data comparability across experiments, saving time and resources by eliminating redundant control tests. This strategy not only streamlines the research process but also improves the reliability of results, making it a sensible, resource-efficient method that ultimately conserves public funding in the pursuit of AD treatments.

Inverse dose protraction effects of low-LET radiation: Evidence and significance.

Biological effects of ionizing radiation vary not merely with total dose but also with temporal dose distribution. Sparing dose protraction effects, in which dose protraction reduces effects of radiation have widely been accepted and generally assumed in radiation protection, particularly for stochastic effects (e.g., solid cancer). In contrast, inverse dose protraction effects (IDPEs) in which dose protraction enhances radiation effects have not been well recognized, nor comprehensively reviewed. Here, we review the current knowledge on IDPEs of low linear energy transfer (LET) radiation. To the best of our knowledge, since 1952, 157 biology, epidemiology or clinical papers have reported IDPEs following external or internal low-LET irradiation with photons (X-rays, γ-rays), β-rays, electrons, protons or helium ions. IDPEs of low-LET radiation have been described for biochemical changes in cell-free macromolecules (DNA, proteins or lipids), DNA damage responses in bacteria and yeasts, DNA damage, cytogenetic changes, neoplastic transformation and cell death in mammalian cell cultures of human, rodent or bovine origin, mutagenesis in silkworms, cytogenetic changes, induction of cancer (solid tumors and leukemia) and non-cancer effects (male sterility, cataracts and diseases of the circulatory system), tumor inactivation and survival in non-human mammals (rodents, rabbits, dogs and pigs), and induction of cancer and non-cancer effects (skin changes and diseases of the circulatory system) in humans. In contrast to a growing body of phenomenological evidence for manifestations of IDPEs, there is limited knowledge on mechanistic underpinnings, but proposed mechanisms involve cell cycle-dependent resensitization and low dose hyper-radiosensitivity. These necessitate continued studies for further mechanistic developments and assessment of implications of scientific evidence for radiation protection (e.g., in terms of a dose rate effectiveness factor).

Inverse dose protraction effects of high-LET radiation: Evidence and significance.

Biological effects of ionizing radiation vary with radiation quality, which is often expressed as the amount of energy deposited per unit length, i.e., linear energy transfer (LET). For acute irradiation, high-LET radiation generally produces greater biological effects than low-LET radiation, but little knowledge exists as to how dose protraction modifies effects. In this regard, inverse dose protraction effects (IDPEs) are phenomena in which dose protraction enhances effects, contrasting with sparing dose protraction effects in which dose protraction reduces effects. Here, we review the current knowledge on IDPEs of high-LET radiation. To the best of our knowledge, since 1967, 80 biology or epidemiology papers have reported IDPEs following external or internal high-LET irradiation with neutrons, deuterons, α-particles, light ions, or heavy ions. IDPEs of high-LET radiation have been described for biochemical changes in cell-free macromolecules, neoplastic transformation, cell death, DNA damage responses and gene expression changes in mammalian cell cultures of human or rodent origin, gene mutations, cytogenetic changes, cancer, non-cancer effects (e.g., testicular effects, cataracts, cardiovascular diseases) and life shortening in non-human mammals (rodents and dogs), and induction of lung cancer and bone tumors in humans. For external irradiation of mammalian cells in vitro and mammals in vivo, IDPEs of low- and high-LET radiation have been reported for radiation doses spanning in excess of three or four orders of magnitude in slightly different ranges, and for radiation dose rates both spanning over six orders of magnitude in different ranges. IDPEs of high-LET radiation in humans have been reported following internal exposure, but not external exposure. Manifestations and mechanisms of IDPEs of high-LET radiation are far less understood than those of low-LET radiation, warranting further studies that will be pivotal to assess the implications for radiation protection.

Application of semi-quantitative loop-mediated isothermal amplification for gene expression study in Expi293 cells

BACKGROUND: Mammalian cell cultures play a key role in the pharmaceutical industry, requiring constant monitoring of the cell conditions during fermentation. In addition to monitoring of the physical-chemical parameters, it is important to evaluate the transcription state of cells, for which gene expression analysis is used. Currently, quantitative reverse transcription polymerase chain reaction (qPCR) is the dominant method. However, the loop-mediated isothermal amplification (LAMP) technique also attracts attention due to its high specificity, sensitivity and reaction rate. LAMP is becoming a promising tool for rapid analysis of gene expression, especially under the conditions of limited biological material or a large volume of samples. AIM: Development of a technique for semi-quantifying the expression level of target genes in human cell culture Expi293 using LAMP. MATERIALS AND METHODS: For LAMP, a recombinant large fragment of Bacillus stearothermophilus DNA polymerase (Bst-pol) was obtained, purified, and optimal reaction conditions were determined. SYBR Green I and LUCS13 were used as intercalating dyes. The amplification parameters for different concentrations of the enzyme and the dye were analyzed. Standard SYBR Green I kits were used for qPCR. Both methods were compared when analyzing the expression of IGF1, FGF2 and EIF3i genes in cell lines with an increased expression level of these genes. RESULTS: It has been shown that using LUCS13 dye provides the classic S-shape of the signal accumulation curve in LAMP, while using SYBR Green I dye causes artifacts. The optimal concentration of Bst-pol was 40 ng/µl. When comparing the two methods, it was found that LAMP has greater sensitivity, allows determining gene expression with an accuracy comparable to qPCR, demonstrating a shorter reaction time (up to 35 minutes). CONCLUSION: Although qPCR remains the main method for assessing the level of gene expression, LAMP offers a number of advantages that make it an attractive alternative for various biotechnological purposes. Due to its high speed, ease of execution and accessibility, as well as high sensitivity and specificity, LAMP is a valuable technique for rapid analysis of gene expression during cell culture monitoring.

Enhancing medicinal proteins production in plant bioreactors: A focal review on promoters.

The use of plant bioreactors for the production of medicinal proteins has emerged as a promising and cost-effective alternative to traditional microbial and mammalian cell culture systems. This review provides a focused examination of the critical role of promoters in enhancing the production of therapeutic proteins within plant-based platforms. We discuss the latest advancements in promoter discovery, modification, and optimization for the expression of medicinal proteins in plants. The review highlights the challenges and opportunities associated with various types of promoters, including constitutive, tissue-specific, and inducible promoters, and their impact on medicinal protein yield and quality. Case studies are presented to illustrate the successful application of these promoter engineering techniques in plant bioreactors, emphasizing the potential for scalable and sustainable production of pharmaceutical proteins. Additionally, we explore the strategies for improving promoter function. This review is intended to guide researchers and industry professionals in the selection and design of promoters for the enhanced production of medicinal proteins in plant bioreactors.

Synthetic Engineering of Cortical Polarity During Mitosis Using Designed Proteins.

During asymmetric cell division, cortical polarity cues drive the polarization of the microtubule cytoskeleton to ensure the generation of two daughter cells with different fates. While this a critical process for development and tissue homeostasis, the underlying molecular mechanisms orchestrating those processes are not completely understood, especially in mammals. Here, we present an assay that allows the study of the molecular mechanisms driving mammalian asymmetric cell division in a high-throughput manner by capitalizing on protein design to engineer cortical polarity of virtually any protein of interest in otherwise unpolarized mammalian culture cells.

RNA programmable cell targeting and manipulation with CellREADR.

Methods that provide specific, easy, and scalable experimental access to animal cell types and cell states will have broad applications in biology and medicine. CellREADR - Cell access through RNA sensing by Endogenous ADAR (adenosine deaminase acting on RNA), is a programmable RNA sensor-actuator technology that couples the detection of a cell-defining RNA to the translation of an effector protein to monitor and manipulate the cell. The CellREADR RNA device consists of a 5' sensor region complementary to a cellular RNA and a 3' payload coding region; payload translation is gated by the removal of a STOP codon in the sensor region upon base pairing with the cognate cellular RNA through an ADAR-mediated A- to-I editing mechanism ubiquitous to metazoan cells. CellREADR thus highlights the potential for RNA-based monitoring and manipulation of animal cells in ways that are simple, versatile, and generalizable across tissues and species. Here, we describe a detailed protocol for implementing CellREADR experiments in cell cultures and in animals. The procedure includes sensor and payload design, cloning, validation and characterization in mammalian cell cultures. The in vivo animal protocol focuses on AAV-based delivery of CellREADR using brain tissue as examples. We describe current best practices, various experimental controls and trouble-shooting steps. Beginning from sensor design, the validation of RNA sensor-actuators in cell cultures can be completed in 2-3 weeks. The construction of AAV vectors and their in vivo validation and characterization will take additional 6-8 weeks.

A data-driven approach for cell culture medium optimization

Cell culture media are critical for cell propagation and bioproduction to be as efficient as possible to meet medical and pharmaceutical requirements. However, optimizing medium composition to achieve optimal cell culture remains a significant challenge due to the complexity of living cells and their required media. This study, which is a significant contribution to the field, addresses the need for data-driven techniques in cell culture technologies by integrating active machine learning (ML) to reformulate a widely used base medium for mammalian cell culture. The optimization process was facilitated by developing various ML models, which accounted for experimental data processing and time consumption. It provided a detailed methodology and essential knowledge for utilizing ML in medium development. Growth determinative medium components were identified through data mining and scale-up culture. In addition, RNA sequencing analysis indicated that active learning finetuned the media for the changes in gene expression for improved cell culture. This study offers new insights and methodologies to be applied to cell culture for future medical purposes.

Kunitz-type trypsin inhibitor from durian (Durio zibethinus) employs a distinct loop for trypsin inhibition.

Kunitz-type trypsin inhibitors are ubiquitous in plants. They have been proposed to be a part of a defense mechanism against herbivores. Trypsin inhibitors also have potential applications in the biotechnology industry, such as in mammalian cell culture. We discovered that durian (Durio zibethinus) seed contains Kunitz-type trypsin inhibitors as identified by N-terminal sequencing and mass spectrometry. Eleven new trypsin inhibitors were cloned. The D. zibethinus trypsin inhibitors (DzTIs) that are likely expressed in the seed were produced as recombinant proteins and tested for trypsin inhibitory activity. Their inhibitory activity and crystal structures are similar to the soybean trypsin inhibitor. Surprisingly, a crystal structure of the complex between DzTI-4, the DzTI with the lowest inhibitory constant, and bovine trypsin revealed that DzTI-4 utilized a novel tryptophan-containing β1-β2 loop to bind trypsin. Site-direct mutagenesis confirmed the inhibitory role of this loop. DzTI-4 was not toxic to the HEK293 cells and could be used in place of the soybean trypsin inhibitor for culturing the cells under serum-free conditions. DzTI-4 was not toxic to mealworms. However, a mixture of DzTIs extracted from durian seed prevented weight gain in mealworms, suggesting that multiple trypsin inhibitors are required to achieve the antinutritional effect. This study highlights the biochemical diversity of the inhibitory mechanism of Kunitz-type trypsin inhibitors and provides clues for further application of these inhibitors.

Convenient production of a novel recombinant antibody against periodontitis biomarker S100A8

S100A8 serves as a biomarker for periodontitis and is involved in inflammatory processes, making its detection highly important. In this study, we produced recombinant 5A11 (r5A11) through mammalian cell culture. By employing a three-step process of transfection, suspension cell culture, and purification, we conveniently produced r5A11 with high yield and purity. The limit of detection for the r5A11-based immunoassay was 1.7 ± 0.2 × 10−1 ng/mL, which was higher than that of the commercially available anti-S100A8 antibody. These findings suggest the potential use of this novel antibody in various research applications and practical approaches for simple and sensitive S100A8 detection.

Unveiling tryptophan dynamics and functions across model organisms via quantitative imaging

BackgroundTryptophan is an essential amino acid involved in critical cellular processes in vertebrates, serving as a precursor for serotonin and kynurenine, which are key neuromodulators to influence neural and immune functions. Systematic and quantitative measurement of tryptophan is vital to understanding these processes.ResultsHere, we utilized a robust and highly responsive green ratiometric indicator for tryptophan (GRIT) to quantitatively measure tryptophan dynamics in bacteria, mitochondria of mammalian cell cultures, human serum, and intact zebrafish. At the cellular scale, these quantitative analyses uncovered differences in tryptophan dynamics across cell types and organelles. At the whole-organism scale, we revealed that inflammation-induced tryptophan concentration increases in zebrafish brain led to elevated serotonin and kynurenine levels, prolonged sleep duration, suggesting a novel metabolic connection between immune response and behavior. Moreover, GRIT’s application in detecting reduced serum tryptophan levels in patients with inflammation symptoms suggests its potential as a high-throughput diagnostic tool.ConclusionsIn summary, this study introduces GRIT as a powerful method for studying tryptophan metabolism and its broader physiological implications, paving the way for new insights into the metabolic regulation of health and disease across multiple biological scales.

Assessing Drug Sensitivity in Fission Yeast Using Half-Maximal Inhibitory Concentration (IC50) Assays.

Fission yeast is an excellent model organism in which to study mammalian drug sensitivities. In addition to building a mechanistic picture of drug effect, fission yeast screens may be valuable in determining compounds that show synthetic lethality effects. While compounds might be screened for a variety of phenotypes, an effective method is to detect the proliferative effects of a new compound on a yeast culture. This is traditionally performed in acute viability assays or spot tests; both methods require some knowledge of concentration to observe an effect. Mammalian cell culture experiments that assess proliferation to indicate drug dose and effect are well described. However, differences between S. pombe growth characteristics and mammalian cells mean that importing a mammalian viability assay requires consideration of potential effects on fission yeast biology. We describe the half-maximum inhibitory (IC50) dose as a method of rapid proliferation effect screening. IC50 determination is performed on liquid cultures in 96-well plates and may be developed for initial compound library uses or in synthetic lethality screening of drug effects.

Experimental arboviral infection of mosquito larvae: A novel approach for vector competence studies

Vector competence studies in mosquitoes present valuable opportunities to explore arboviral transmission and virus-vector interactions. However, oral infection studies in mosquitoes can be challenging. An alternative approach is to infect mosquitoes during their aquatic larval stage, resulting in the emergence of infected adults. To investigate the potential of this method, Culex pipiens biotype molestus larvae were infected with Usutu virus (USUV, Orthoflavivirus usutuense). For this purpose, larvae were exposed to USUV-infected mammalian and mosquito cell cultures for 24 h before being reared to adults. Subsequent analysis via RT-qPCR revealed that the Culex larvae successfully acquired USUV from the infected cells and exhibited high susceptibility to infection. Immediately after emergence, 32.10 % (26/81) of male and 41.03 % (16/39) of female mosquitoes tested positive for USUV RNA. Notably, females that were incubated for 15 days post-emergence demonstrated even higher infection rates, reaching 100.00 % (23/23). In addition, viral RNA and infectious particles were detected in some saliva samples, indicating the potential for transmission. This experimental infection of mosquito larvae thus offers the opportunity to produce infected adult mosquitoes for studies enhancing our understanding of virus-vector interactions, co-infections, and transmission routes. Such research contributes to better public health strategies addressing arboviral diseases.

Comparative analysis of canine and human HtrA2 to delineate its role in apoptosis and cancer.

Therapeutically, targeting the pro- and anti-apoptotic proteins has been one of the major approaches behind devising strategies to combat associated diseases. Human high-temperature requirement serine protease A2 (hHtrA2), which induces apoptosis through both caspase-dependent and independent pathways is implicated in several diseases including cancer, ischemic heart diseases, and neurodegeneration, thus making it a promising target molecule. In the recent past, the canine model has gained prominence in the understanding of human pathophysiology that was otherwise limited to the rodent system. Moreover, canine models in cancer research provide an opportunity to study spontaneous tumors as their size, lifespan, and environmental exposure are significantly closer to that of humans compared with laboratory rodents. Therefore, using HtrA2 as a model protein, comparative analysis has been done to revisit the hypothesis that canines might be excellent models for cancer research. We have performed evolutionary phylogenetic analyses that confirm a close relationship between canine and human HtrA2s. Molecular modeling demonstrates structural similarities including orientation of the catalytic triad residues, followed by in silico docking and molecular dynamics simulation studies that identify the potential interacting partners for canine HtrA2 (cHtrA2). In vitro biophysical and protease studies depict similarities in interaction with their respective substrates as well as transient transfection of cHtrA2 in mammalian cell culture shows induction of apoptosis. This work, therefore, promises to open a new avenue in cancer research through the study of spontaneous cancer model systems in canines.