Animal testing has shaped modern drug development for decades, but its limitations become especially visible when scientists evaluate cancer immunotherapies. These treatments are designed to stimulate the immune system to recognize and destroy malignant cells, yet the same powerful immune activation can trigger life-threatening complications in patients. Researchers at the University of Rochester are developing a human-cell-based alternative: miniature tissue models known as organs-on-a-chip that could help predict these toxicities before experimental medicines reach clinical trials.

The work is being led through the university’s Translational Center for Barrier Microphysiological Systems, or TraCe-bMPS. The center is building drug-development tools around modular µSiM tissue chips, devices that contain ultrathin membranes populated with human cells. Unlike conventional laboratory cultures, these systems are engineered to reproduce key features of biological barriers, including the physical separation between tissues, controlled exposure to drugs, and measurable inflammatory responses. Their modular construction also allows the devices to be manufactured in large quantities with consistent dimensions and materials.

The platform is based primarily on acrylic components and incorporates membranes designed to mimic interfaces found in the human body. Such barriers regulate the movement of molecules and immune cells between compartments, making them central to drug absorption, toxicity, and inflammation. By assembling reproducible chips with human cells, researchers can study how a therapy affects a tissue under conditions that more closely approximate human biology than many animal models. The design also makes it possible to modify individual components for different organs or disease settings.

A major feature of the Rochester system is the integration of sensors directly into the chips. Developed in part through the work of biomedical engineering professor Benjamin Miller, these sensors can track barrier function and inflammatory signaling in real time. Instead of waiting until the end of an experiment to examine damaged cells, researchers can observe how a tissue responds as a drug is introduced. Changes in electrical or molecular signals may reveal that a barrier is becoming more permeable or that inflammatory pathways are being activated, providing an early warning of toxicity.

The immediate focus is on two severe complications associated with some cancer immunotherapies. Cytokine release syndrome, commonly called CRS, occurs when activated immune cells release large quantities of signaling proteins known as cytokines. The resulting systemic inflammation can cause fever, low blood pressure, breathing difficulties, and, in the most serious cases, organ failure. Immune effector cell-associated neurotoxicity syndrome, or ICANS, can affect the nervous system, producing confusion, seizures, impaired speech, or other neurological symptoms. Both conditions are difficult to predict reliably with animal testing because species differ in immune signaling networks and cell-surface receptors.

“The goal is to predict these toxicities from human cells on a chip, before a drug ever reaches a patient, and to do it without relying on animal models that have repeatedly failed to predict CRS in people,” says James McGrath, director of TraCe-bMPS and the William R. Kenan Jr. Professor of Biomedical Engineering. The approach does not simply attempt to reproduce a whole human body in miniature. Instead, it focuses on specific biological barriers and immune interactions that are directly relevant to the safety question being studied.

The project has now reached an important regulatory stage. The team was accepted into the US Food and Drug Administration’s Innovative Science and Technology Approaches for New Drugs, or ISTAND, pilot program. ISTAND is intended to help evaluate emerging methods that could improve drug development and eventually support regulatory decisions. Acceptance does not mean that the tissue-chip platform has already been approved for routine use, but it creates a formal pathway for the researchers to work with the FDA on defining the evidence needed for qualification.

The Rochester group is preparing a detailed qualification plan that will describe clinical considerations, timelines, data-sharing procedures, and statistical methods for evaluating the technology. If the platform ultimately receives FDA qualification, pharmaceutical companies could use its results as part of the evidence submitted in support of new drug applications. Such recognition would represent a significant step for organs-on-a-chip research, which has expanded rapidly but has only rarely progressed into formal regulatory programs.

The effort comes as US regulators seek alternatives to animal testing in preclinical safety studies. The FDA Modernization Act 2.0, enacted in 2022, opened the door for non-animal methods, while the agency’s 2025 roadmap outlined further efforts to reduce reliance on animal models. Researchers say pharmaceutical companies are showing strong interest because many immunotherapies are already in clinical development and carry risks of CRS or ICANS. The Rochester team collaborated with Graham Marsh of the Critical Path Institute and consulted with scientists at Pfizer about how the tool could fit into real-world drug development. If successful, the technology could give researchers a more human-relevant way to identify dangerous immune reactions early, helping developers eliminate unsafe candidates and refine promising treatments before patients are exposed.

Subject of Research: Human-cell tissue chips for predicting cancer immunotherapy toxicities.

Web References: University of Rochester; Translational Center for Barrier Microphysiological Systems; University of Rochester Biomedical Engineering; FDA ISTAND pilot program; FDA Modernization Act 2.0.

Image Credits: URochester photo / J. Adam Fenster.

Keywords: Drug discovery, tissue chips, organs-on-a-chip, cancer immunotherapy, cytokine release syndrome, CRS, ICANS, neurotoxicity, animal testing alternatives, biomedical engineering, microphysiological systems, human-cell models.

Cancer researchers have reported a new strategy that combines bacterial tumor targeting, nutrient deprivation and immune stimulation in a single therapeutic design. The approach uses an engineered conjugate built from L-asparaginase and flagellin, linked to a system that directs the treatment toward calreticulin-bearing cancer cells. In experiments involving Salmonella-mediated tumor therapy, the conjugate enhanced antitumor activity compared with bacterial treatment alone, according to a study published in Cell Death Discovery.

The work addresses a long-standing challenge in cancer therapy: how to make powerful treatments concentrate inside tumors while limiting damage to healthy tissues. Attenuated strains of Salmonella have attracted interest because they can preferentially accumulate in the abnormal environment of solid tumors. Tumors often contain regions with poor oxygen levels, disorganized blood vessels and local immune suppression, conditions that can support bacterial growth. Once inside these sites, therapeutic Salmonella can act as a biological delivery platform and stimulate immune responses against malignant cells.

The researchers focused on calreticulin, a protein normally found inside the endoplasmic reticulum, where it helps regulate calcium storage and protein folding. Under cellular stress, including stress caused by chemotherapy, radiation or other anticancer treatments, calreticulin can move to the outer surface of a cancer cell. There, it functions as an “eat-me” signal, alerting immune cells that the damaged cell should be engulfed. Because surface-exposed calreticulin is associated with immunogenic forms of cell death, it provides a potential molecular address for directing therapeutic agents toward stressed tumor cells.

The experimental construct combines this targeting concept with L-asparaginase, an enzyme already used in clinical oncology, especially in the treatment of acute lymphoblastic leukemia. L-asparaginase breaks down circulating L-asparagine into aspartic acid and ammonia. Some cancer cells, particularly those with limited capacity to synthesize their own asparagine, depend heavily on the amino acid supplied through the bloodstream. Depleting extracellular asparagine can therefore interrupt protein production, trigger metabolic stress and promote cancer-cell death. The enzyme’s effectiveness, however, can be limited by immune reactions, pharmacological instability and toxicity, making targeted delivery an important goal.

The second component, flagellin, is the structural protein that forms the filament of bacterial flagella. It is also a potent molecular signal for the innate immune system. Immune cells recognize flagellin primarily through Toll-like receptor 5, while intracellular sensing pathways can activate inflammasome components such as NLRC4. These signals can promote the release of inflammatory mediators, stimulate antigen-presenting cells and help convert an immunologically “cold” tumor into one more visible to the immune system. By incorporating flagellin into the therapeutic design, the researchers sought to make the treatment not only directly toxic to tumor cells but also capable of amplifying antitumor immunity.

The study’s central finding was that the calreticulin-targeting L-asparaginase–flagellin conjugate strengthened the antitumor effects of Salmonella-based therapy. Rather than relying on a single mechanism, the treatment brings together several forms of pressure on the tumor. Salmonella can concentrate within the tumor microenvironment, the targeting component can help associate the conjugate with calreticulin-exposing cancer cells, L-asparaginase can deprive vulnerable cells of an essential nutrient, and flagellin can activate immune surveillance. The resulting combination is designed to produce a chain reaction in which metabolic stress and immune stimulation reinforce one another.

This type of combination may be particularly valuable because tumors frequently adapt when exposed to one therapeutic pressure. A cancer cell that survives nutrient deprivation may still be eliminated if immune recognition is intensified. Likewise, an immune response that is too weak to control a tumor may become more effective when bacterial localization and enzyme-mediated damage increase the number of abnormal antigens and danger signals released by dying cells. The researchers’ findings suggest that coordinating these mechanisms can improve the performance of bacteria-assisted cancer treatment in experimental settings.

The approach also reflects a broader shift in cancer research toward programmable biological medicines. Instead of treating bacteria only as infectious threats, scientists are redesigning them as localized delivery vehicles capable of carrying enzymes, immune activators or molecular probes. The advantage is spatial: a therapeutic payload can be produced or concentrated near the tumor rather than distributed uniformly throughout the body. The challenge is equally significant. Any clinical version would need precise control over bacterial attenuation, immune activation, enzyme exposure and potential inflammation, while also demonstrating reliable performance across genetically diverse tumors.

Calreticulin targeting may provide a useful way to address some of that complexity because the protein’s appearance on the cell surface is linked to cellular stress and treatment response. However, the extent and duration of calreticulin exposure can vary between tumor types and individual patients. Future studies will need to determine which cancers are most suitable for this strategy, how calreticulin levels predict treatment response and whether the conjugate can be combined safely with established immunotherapies such as immune-checkpoint inhibitors. Researchers will also need to assess pharmacology, manufacturing consistency and the possibility of immune reactions against the bacterial or enzymatic components.

The findings position the engineered conjugate as a promising experimental platform rather than an immediately available therapy. By merging tumor-homing bacteria with a calreticulin-directed enzyme and an innate immune stimulant, the study illustrates how cancer treatments can be designed to attack malignant cells on multiple biological fronts. If the results are confirmed in further preclinical testing and eventually in carefully controlled clinical trials, this strategy could help turn Salmonella from a passive carrier into an active, multifunctional partner in cancer immunotherapy.

Subject of Research: Calreticulin-targeted L-asparaginase–flagellin conjugate used with Salmonella-mediated cancer therapy.

Article Title: Calreticulin-targeting L-asparaginase-flagellin conjugate enhances Salmonella-mediated antitumor efficacy.

Article References: Nguyen, DH., Afzal, A.R., Nguyen, P.TM. et al. Calreticulin-targeting L-asparaginase-flagellin conjugate enhances Salmonella-mediated antitumor efficacy. Cell Death Discov. (2026). https://doi.org/10.1038/s41420-026-03300-x

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41420-026-03300-x

Keywords: cancer immunotherapy, Salmonella, calreticulin, L-asparaginase, flagellin, tumor targeting, bacterial therapy, antitumor efficacy, immunogenic cell death