1. A patient-derived colorectal cancer organ-on-chip model for functionally interrogating cancer-associated fibroblast heterogeneity in tumor invasion

Bethany Haliday — University of Southern California

The tumor microenvironment (TME) has become a major area of study made possible by single cell and spatial -omics revealing significant interactions between the tumor cells and the surrounding immune and stromal cells. Cancer associated fibroblasts (CAFs) make up a majority of the stroma and are associated with disease progression, metastasis, and drug resistance. However, CAFs are very heterogenous within the TME with several reported subtypes, including inflammatory CAFs (iCAFs) and myofibroblast-like CAFs (myCAFs). The functions of these CAF subtypes, specifically in the context of primary tumor invasion, are largely unknown beyond -omics based studies. With metastatic disease having a particularly poor prognosis, it is vital to elucidate the functions of these CAF subtypes to better understand and ultimately target CAF-induced tumor progression.

To address this gap, we built a diverse biobank of CRC patient-derived tissue samples and matched CAF lines. Single cell RNA-seq of patient tissues confirmed distinct CAF subpopulations with expression patterns consistent with iCAF and myCAF profiles. These gene signatures were then applied to bulk RNA-sequencing of the patient-derived CAF lines and revealed that each line trended towards a distinct CAF subtype. Flow cytometry and secretome analysis further confirmed expression of subtype-specific markers, suggesting that each CAF line retains features of the originating subtypes.

To investigate how these different CAFs influence tumor cell invasion, we developed a CRC organ-on-chip model. Specifically, this microfluidic device simulates gut-relevant biomechanical cues, including fluid flow and peristalsis-like motion, and is composed of two extracellular matrix-coated overlapping channels separated by a porous membrane, allowing biochemical and cellular exchanges between compartments. Patient-derived CAFs and GFP-labelled CRC tumor cells are cultured together in the top “epithelial” channel, while endothelial cells line the bottom “vascular” channel. Using confocal microscopy, GFP+ tumor cells intravasating into the vascular channel are tracked and quantified, providing real time invasion rates. When exposing human CRC cells on-chip to CAF-conditioned media and in physical co-cultures, we observed differences in CAF-induced CRC invasion. Interestingly, a myCAF-enriched line induced the highest invasion rates in co-culture conditions and invaded into the vascular channel preceding the tumor cells, suggesting contact-dependent signaling and extracellular matrix remodeling. Collectively, these results show that the CRC-on-chip platform can distinguish the functional phenotypes of different CAF subtypes during primary tumor invasion. Next, we plan to utilize this platform to study the mechanisms driving these subtype-specific behaviors, through both genetic alterations and drug perturbations.

2. Design and Evaluation of CNS-Targeted Antibodies in a Humanized Mouse Model of Microglial HIV infection

Catherine Diadhiou — University of Southern California

HIV persists in the body despite antiretroviral therapy (ART) by establishing long-lived latent reservoirs, including in the central nervous system (CNS). Microglia, the brain’s resident immune cells, harbor this latent virus and contribute to HIV-associated neurocognitive disorders (HAND). Because HIV does not infect mouse cells, “humanized” mice, containing human immune cells, are required to study HIV infection and evaluate targeted interventions. Broadly neutralizing antibodies (bnAbs) represent a promising alternative therapy that could potentially target a persistent CNS reservoir of HIV. However, it is challenging to deliver bnAbs to the CNS, and less than 0.1% of antibody plasma concentration reaches the CNS. Our goal is to engineer anti-HIV antibodies that better cross the blood-brain barrier (BBB) by exploiting receptor mediated transcytosis (RMT).

We use a humanized IL34-NOG mouse model engrafted with human hematopoietic stem and progenitor cells (HSPCs). This strain is transgenic for the human IL34 cytokine, which supports microglial development and enables robust reconstitution of human microglia in the CNS. Single-cell RNA sequencing demonstrates that these microglia exhibit transcriptional profiles consistent with mature human microglia, supporting the physiological relevance of this system. Beyond HIV, this model provides a versatile platform for studying human microglial biology in neurodegenerative diseases.

We subsequently developed prototype brain-penetrating antibodies (BP-Abs) by combining the anti-HIV VHH domain, J3, with different RMT receptor ligands (aTfR, aTMEM30A, aLRP1) that cross the BBB. We screened the BP-Ab candidates in an in vitro BBB model. We also enhanced its in vivo circulatory half-life and ultimate CNS penetration levels by adding an anti-albumin VHH domain (aAlb). Our top candidate, J3-aAlb-aTfR, was expressed as a recombinant protein and evaluated for CNS delivery in HIV-infected humanized IL34-NOG mice after i.v. injection, and for its ability to neutralize HIV in vitro.

Our findings by ELISA and immunofluorescence staining showed that recombinant J3-aAlb-aTfR is delivered to the brain and colocalizes with human microglia in the infected mice. These findings highlight that latent CNS HIV reservoirs can be targeted with proper antibody design. This model provides a powerful platform for studying adjunctive CNS targeted strategies to prevent HIV spread in the CNS and mitigate HAND.

3. Fasting-refeeding promotes epithelial regeneration after cytotoxic injury and modulates ISG responses

Yashika Bopanna — Cedars-Sinai Medical Center

The intestinal epithelium continuously renews and rapidly regenerates following injury, processes that are highly responsive to nutrient availability. Nutritional status is a modifiable factor with potential to influence barrier restoration and recovery; however, how dynamic nutrient shifts, including fasting and refeeding, regulate intestinal regeneration remains unclear. We hypothesized that fasting-refeeding modulates epithelial damage and its niche responses to promote repair. We performed a time-course analysis of clinically relevant 5-fluorouracil (5-FU)-induced intestinal injury under ad libitum, fasting, and refeeding conditions. On day 3 post-injury, fasting-refeeding significantly increased epithelial proliferation, indicating a robust diet-dependent regenerative response. Flow cytometry showed expanded SCA-1⁺ epithelial populations in fasted and refed mice. RNA sequencing identified a fasting-associated damage state enriched for interferon-stimulated genes that declined after refeeding, indicating a transient stress response. These findings led us to investigate whether 5-FU-induced DNA damage or fasting-associated metabolic stress activates cGAS–STING signaling to drive the interferon program. Ongoing temporal transcriptional profiling will determine how this response is altered by refeeding and may identify previously unrecognized pathways through which dietary cues regulate the epithelium and its niche during regeneration. Defining these mechanisms will inform dietary or metabolic strategies to improve sustained intestinal recovery following cytotoxic injury.

4. Tracking Clonal Evolution in Prostate Cancer Liver Metastasis Using Longitudinal Acoustic Reporter Gene Imaging

Helya Bavaghar — City of Hope

Background: Liver metastasis is associated with the poorest survival among major metastatic sites in advanced prostate cancer, yet the mechanisms underlying this aggressive phenotype remain poorly understood. We hypothesize that the specialized hepatic microenvironment creates a selective bottleneck favoring particular tumor populations and that radiotherapy imposes additional selection that further reshapes clonal composition. Testing this requires more than endpoint analysis: tumors with similar final sizes may have undergone fundamentally different growth and treatment-response trajectories. We therefore aim to longitudinally track individual metastases and link their behavior to clonal identity.

Methods & Results: To enable this, we engineered murine RM9-ffLuc and human PC3 and DU145 prostate cancer cells with a two-vector, doxycycline-inducible acoustic reporter gene (ARG) system coupled to GFP. ARGs encode gas vesicles (GVs), gas-filled microbial protein nanostructures that generate ultrasound contrast. Following doxycycline induction, stringent FACS gating isolated the highest-expressing fractions (10.1% of RM9, 19.9% of PC3, and 20.5% of DU145 cells), which recovered and expanded in culture. We then evaluated whether engineered cells retained tumorigenicity and reporter function in vivo. RM9-ffLuc-GV cells formed tumors in immunocompetent mice and, following harvest at ~1.5 cm, retained GFP expression and yielded viable reporter-positive cells for clonal expansion. Human PC3-GV and DU145-GV cells similarly formed tumors reaching 549 and 621 mm³, respectively. Finally, PC3-GV cells generated spatially localized xAM ultrasound contrast that co-registered with the cell sample. During destructive imaging, acoustic signal decreased >95% from peak to near-background levels after GV collapse, while B-mode remained stable, confirming GV-specific acoustic contrast.

Conclusion: Together, these preliminary studies establish a functional prostate cancer ARG platform that persists through in vivo tumor growth, permits recovery for downstream clonal analysis, and generates robust acoustic contrast. Integrating this platform with genetic perturbation and clonal barcoding will directly link metastatic trajectories to clonal identity, revealing the mechanisms driving hepatic fitness and radioresistance.