Abstract There is a critical need to develop novel therapeutic strategies and diagnostic tools to precisely deliver treatments to improve survival for men with prostate cancer. To support this development, improved strategies are needed to better understand heterogenous tumor microenvironments and tumor biology that associate with variable treatment responses. We hypothesized that the tumor immune microenvironment (TIME) plays a critical role in treatment resistance. In this study we aimed to evaluate TIME signatures of treatment response and resistance utilizing a novel, integrated technological tool to identify response patterns and enable precision sampling for comparative cellular and molecular analysis. 30 patients with newly diagnosed, locally advanced, high-risk, primary prostate cancer underwent 18F-DCFPyL PSMA PET/MRI with multiparametric MRI scans followed by 3 cycles of chemohormonal therapy (NCT03358563). Repeat PSMA PET/MRI was performed prior to prostatectomy and scans were used to categorize lesions as complete response (CR), partial response (PR), no response (NR) or normal tissue. MRI scans were used to print a 3D mold of the prostate to allow microdissection of regions of interest from the resected prostate. Cellular infiltrates were analyzed by flow cytometry in 3 to 5 tissue specimens per patient. Statistical analysis was performed with One-way ANOVA with Tukey-correction. The frequency of CD8+ T cells in the total CD45+ infiltrate was highest in normal and CR areas and was significantly reduced in PR vs CR (p<0.01). CXCR3+CD8+ and CD103+CD8+ T cell frequencies were also reduced in PR vs CR foci (p<0.01, p<0.05, respectively). Meanwhile, the frequency of CXCR3+CD8+ T cells was highest and significantly elevated in CR vs normal tissue. A tendency of reduced CCR6+, CXCR5+, and CCR4+CD8+ T cells was observed in PR vs CR foci, while those frequencies remained higher in normal and CR areas. An increase in total CD8+ and CD103+CD8+ T cells associated with longer progression-free survival. Additionally, the analysis of EpCAM+ cells showed a significant increase in B7H3 expression in PR vs CR lesions (p<0.05) and a tendency of reduced HLA I expression in foci that associated with treatment resistance. We are currently integrating analysis of myeloid cells and transcriptomic analysis of sorted CD4+, CD8+ and CD11b/CD14+ cells to further dissect patterns of therapeutic response in our study cohort. In conclusion, PSMA/PET MRI based precision sampling of tumor tissue associated with differential therapeutic response patterns captured differences in the TIME infiltrates and these observations may provide hypothesis to test biological mechanisms to expedite discovery of targetable mechanisms to improve tumor stratification and targeting in high-risk prostate cancer. Citation Format: Erika Heninger, Jamie M. Sperger, Kristin Weinstein, Brian P. Johnson, Peter G. Geiger, Shane Wells, Steve Y. Cho, Wei Huang, Philippos Tsourkas, Sean McIlwain, Irene M. Ong, Sheena C. Kerr, David F. Jarrard, David J. Beebe, Joshua M. Lang. Differential patterns of immune infiltration in the tumor immune microenvironment associate with therapeutic response in primary prostate cancer following chemohormonal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1175.

Clear cell renal cell carcinoma (ccRCC) exhibits heterogeneity in immune infiltration and clinical outcomes, but the mechanisms governing recruitment and organization of tumor-reactive CD8+ T cells remain incompletely defined. We investigated the role of the CXCL13-CXCR5 axis in shaping CD8+ T cell recruitment, differentiation, and immune organization in high-risk, non-metastatic ccRCC. Human tumor, plasma, and matched adjacent kidney specimens were analyzed using ELISA, quantitative PCR, migration assays, multiplex immunofluorescence, single-cell RNA sequencing, spatial transcriptomics, and a syngeneic mouse model. CXCL13 was among the most upregulated chemokines in ccRCC relative to matched normal kidney and was embedded within a CD8+ T cell-associated inflammatory transcriptional program. In transwell and microphysiological system (MPS) assays, CXCL13 promoted CD8+ T cell migration, enriched CXCR5+ cells among migrating CD8+ T cells and showed reduced migration after CXCL13 or CXCR5 blockade. Single-cell analyses identified CXCR5 expression within stem-like CD8+ T cell states associated with TCF7 and IL7R, whereas CXCL13 associated with later cytotoxic/exhausted states along a continuous differentiation landscape. Spatial transcriptomics demonstrated that stem-like CD8+ T cells localized within structured lymphoid aggregates enriched for B cells, coordinated CXCL13/CXCR5 expression, and signaling programs. In vivo, tumor-derived CXCL13 suppressed tumor growth, increased intratumoral CD8+ T cell infiltration, and enriched CXCR5+TCF1+CD8+ stem-like T cells. In human tumors, higher CXCL13 expression correlated with increased CXCR5+CD8+ T cell infiltration and improved recurrence-free survival. These findings identify CXCL13 as a regulator of immune recruitment and niche organization and support the CXCL13-CXCR5 axis as a biomarker and possible therapeutic target in ccRCC.

Exclusive Liquid Repellency Isolation (ELRi) is a novel, chip-free platform designed for efficient microscale immune cell isolation. Leveraging the inherent exclusive liquid repellency (ELR) properties of polypropylene tubes with an oil overlay, ELRi ensures that small sample volumes, as low as 8 μL, have no contact with container walls, preventing cell loss. Here we describe ELRi and demonstrate magnetic bead-based isolation of diverse immune cells, including T cells, and monocytes, from whole blood. The platform's utility is highlighted in its application to pediatric asthma research, where sample volume is highly restricted. It has been reported that the T cell homing receptor CCR7 is downregulated in cells from asthmatic patients, but a direct functional link to impaired cell migration remains unconfirmed (1, 2). Using ELRi-isolated cells, we provide the first functional evidence that T cells from asthmatic children exhibit significantly impaired chemotaxis toward the CCR7 ligand, CCL21. By simplifying RBC depletion and enabling such functional assays alongside RNA sequencing from the same tiny sample, ELRi overcomes the limitations of large-volume flow cytometry or cell-specific sorting methods. Easily integrated into lab workflows and scalable for various needs, ELRi facilitates more frequent, minimally invasive, and functionally informative immune profiling of restricted volume samples.

Head and neck cancer (HNC) is the 6th most common malignancy worldwide. 60% of patients present with advanced disease and approximately 50% of patients recur following primary treatment. Chemoradiation remains a standard of care for most patients. However, clinicians lack functional tools to predict which patients will respond to chemoradiation prior to treatment and current models, including organoids and animal model systems, fail to capture either full complexity or patient-to-patient heterogeneity of the individual HNC tumor and microenvironment (TME). Here, we have developed, characterized, and tested a patient-specific microphysiological system (MPS) that reconstructs the HNC TME in a vascularized 3D environment. This MPS was constructed from malignant cells, fibroblasts, and immune cells from a patient's surgically resected tumor, seeded within a 3D hydrogel with molded endothelial lumens. Single-cell RNA sequencing confirmed that the MPS preserved 12 transcriptionally distinct cell populations found in matched native tissue. The platform recapitulated tumor hypoxia, with a 12-fold increase in hypoxic marker expression that altered radiation response, consistent with clinical HNC biology. Compartment-resolved imaging revealed distinct treatment dynamics in tumor, stromal, and vascular regions, and individual patients exhibited divergent responses to chemoradiation in spheroid morphology, cell viability, and migration. We found the slope of spheroid area change with treatment tracked with tumor recurrence, suggesting this metric could serve as a functional predictor of therapeutic response.

Early skin inflammation requires coordinated immune regulation, with neutrophils acting as first-line responders. While the blood vasculature and its role in neutrophil recruitment during infection has been extensively studied, the lymphatic system remains comparatively understudied despite its known role in immune cell trafficking. Growing evidence suggests lymphatic vessels actively participate in regulating inflammatory responses, yet whether they coordinate neutrophil behavior during skin infection remains unclear. Staphylococcus aureus is particularly problematic in this context, employing multiple immune evasion strategies and representing a major driver of antibiotic-resistant skin and soft tissue infections worldwide. To address this gap, we developed a human-based 3D microphysiological system incorporating luminal lymphatic endothelial vessels, a collagen matrix and bacteria to model an infected microenvironment. We evaluated neutrophil migration, phagocytosis and NETosis in response to Escherichia coli and S. aureus. Lymphatic endothelium amplified neutrophil migration in a bacterial-dependent manner, with E. coli promoting directional migration toward the vessel while S. aureus suppressed migration and directionality despite increased phagocytic uptake. S. aureus also induced myeloperoxidase-positive NETs with nuclear morphology consistent with vital NETosis, rescued by DNase treatment. To our knowledge, this is the first demonstration that lymphatic endothelium directly drives neutrophil behavior during skin infection.

There is a critical need to develop novel therapeutic strategies and diagnostic tools to precisely deliver treatments to improve survival for men with prostate cancer (PCa). To support this development, improved strategies are needed to better understand heterogenous tumor microenvironments and tumor biology that associate with variable treatment responses. We hypothesized that the tumor immune microenvironment (TIME) plays a critical role in treatment resistance. In this study we aimed to evaluate TIME signatures of treatment response and resistance utilizing a novel, integrated technological tool to identify response patterns and enable precision sampling for comparative cellular and molecular analysis. 30 patients with newly-diagnosed, locally advanced, high-risk, primary PCa underwent 18F-DCFPyL PSMA PET with multiparametric MRI (mpMRI) imaging on a dedicated PET/MRI scanner followed by 3 cycles of chemohormonal therapy (NCT03358563). Repeat PSMA PET/MRI was performed prior to prostatectomy and scans were interpreted by an experienced radiologist and nuclear medicine physician as complete response (CR), partial response (PR), no response (NR) or normal tissue. MRI scans were used to print a 3D mold of the prostate to allow PET and MRI directed mapping and microdissection of regions of interest from the resected prostate with slice-by-slice and lesion-to-lesion correlation. Cellular infiltrates were analyzed by flow cytometry in 3 to 5 tissue specimens per patient. The frequency of CD8+ T cells in the total CD45+ infiltrate was highest in normal and CR areas and was significantly reduced in PR vs CR (p<0.01). CXCR3+CD8+ and CD103+CD8+ Tcell frequencies were also reduced in PR vs CR foci (p<0.01, p<0.05, respectively). Meanwhile, the frequency of CXCR3+CD8+ T and CXCR3+++CD8+ T cells was highest and significantly elevated in CR vs normal tissue suggesting enrichment of activated, homing, Tc1 CD8T cells. An increase in total CD8+ and CD103+CD8+ T cells was associated with longer progression-free survival. DESeq2 analysis of bulk mRNA sequencing showed enrichment of CD8a and ITGAE (CD103) gene expression differential in CR vs PR lesions (p=0.028, p=0.00029, respectively). CD8a expression was reduced in panCK- AOI of resistant (PR) foci by GeoMx WTA spatial transcriptomic analysis in two model patients with multi-focal tumors. Flow analysis of EpCAM+ cells had a significant increase in B7H3 expression in PR vs CR lesions (p<0.05). We are currently integrating analysis of myeloid cells and expand spatial transcriptomic analysis of matched multi-focal tumors to further dissect patterns of therapeutic response in our study cohort. In conclusion, PSMA /PET and mpMRI based precision sampling of tumor tissue associated with differential therapeutic response patterns captured differences in the TIME infiltrates and these observations may provide hypothesis to test biological mechanisms to expedite discovery of targetable mechanisms to improve tumor stratification and targeting in high-risk prostate cancer. Erika Heninger, Jamie M. Sperger, Kristin Weinstein, Brian P. Johnson, Peter G. Geiger, Shane A. Wells, Steve Y. Cho, Wei Huang, Philippos Tsourkas, Sean McIlwain, Irene M. Ong, David Quigley, David F. Jarrard, Sheena C. Kerr, David J. Beebe, Joshua M. Lang. Differential Patterns of Immune Infiltration in the Tumor Immune Microenvironment Associate with Therapeutic Response in Primary Prostate Cancer Following Chemohormonal Therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A023.

It is well established that the tumor microenvironment (TME) plays a dynamic role in breast cancer progression. Stromal components of the TME, including fibroblasts and the extracellular matrix (ECM), provide biophysical and biochemical cues to drive tumor cell proliferation and invasion. However, how the interplay between activated fibroblast (CAF) subtypes and ECM composition impinges on breast cancer cell behavior is still not well understood. To address this gap, we leveraged microfluidic technologies to engineer a model of the breast TME that incorporates tumor cells, CAFs, and ECM components. Our model consists of a tubular duct filled with MDA-MB-231 or MCF-7 cells surrounded by a 3D collagen type I matrix with or without two human fibroblasts/CAFs. To further investigate how ECM composition affects the stromal compartment, we altered the matrix to include laminin-111 (LN) and thrombospondin-1 (TSP-1), which we previously found to be dysregulated in invasive ductal carcinoma compared to normal breast tissue. By incorporating LN or TSP-1 into the collagen matrix, we identified significant differences in collagen fiber metrics (fiber length, density, and width), matrix stiffness, and alterations in matrix remodeling by CAFs. Specifically, remodeling was more pronounced in LN- vs. TSP-1-enriched microenvironments populated by myofibroblast-like (myCAF-like) cells compared to inflammatory CAFs (iCAF-like). Notably, MDA-MB-231 cell proliferation and invasion were significantly enhanced under these same conditions, and invasion was dependent on the LN-binding receptor, α6-integrin.

Efficient immune cell migration requires physical interactions with surrounding tissues. While tissue matrix mechanics influence leukocyte motility, it is unknown how leukocytes exert pushing and pulling forces to traverse tightly adherent epithelial tissues, which comprise a majority of tissue volume in vivo. Here, we leverage the optical transparency of larval zebrafish to identify how physical interactions with epithelial cells regulate mechanisms of neutrophil force generation to navigate cell-dense tissues. Confining forces from epithelial cells induce a mechanosensitive central actin network, mediated by Cdc42 and WASP, which exerts expansile forces on surrounding cells to dilate a path for migration. In concert, direct cell-to-cell (leukocyte-epithelial) contacts, mediated by integrin ɑE binding to epithelial cadherin, generate tractional forces to enable forward motility. Together, our findings identify how physical interactions with surrounding epithelial cells regulate leukocyte motility through cell-dense tissues in vivo.

Prostate cancer (PCa) is the second leading cause of cancer-related death among men, primarily driven by hormonal mechanisms. First-line treatments include androgen deprivation therapy and androgen receptor pathway inhibitors (ARPIs); however, resistance to these treatments often leads to castration-resistant prostate cancer. Cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) significantly influence treatment resistance; however, their interaction with tumor cells remains poorly understood due to a lack of suitable model systems. We hypothesized that CAFs reduce the cytotoxic effects of docetaxel chemotherapy and the ARPI darolutamide. To investigate this, we established two 3D co-culture systems: STACKs and LumeNEXT allowing us to examine treatment outcomes in the context of the PCa TME. STACKs enables high-throughput assessments, while LumeNEXT recapitulates the TME architecture, including engineered blood vessel mimics to study vascular and epithelial interactions involved in metastasis. We treated androgen-sensitive (LNCaP, LAPC4) and androgen-resistant (22Rv1) tumor spheroids with 10 μM darolutamide or 20 nM docetaxel, in the presence or absence of immortalized CAFs (hPrCSC-44) or normal myofibroblasts (WPMY-1) within the STACKs system. Cytotoxicity was assessed using confocal microscopy. Ongoing work aims to enhance the model by incorporating patient-derived fibroblasts from PCa patients. We characterized primary fibroblasts through bulk RNA sequencing and histological analysis and optimized co-culture conditions before introducing primary CAFs into the LumeNEXT model. Additionally, we included new genetically engineered tumor cell lines (p53/Rb1 double knockout, wild-type, and APIPC) that represent common mutations found in PCa. To measure cell death, we calculated the ratio of Ethidium homodimer+ and Hoechst+ nucleated cells to the total number of Hoechst+ cells in spheroids (≥100 µm). Statistical analyses were conducted using a two-way ANOVA and Tukey's multiple comparisons test. In the STACKs, CAFs significantly reduced docetaxel toxicity in LAPC4, LNCaP, and 22Rv1 compared to conditions without CAFs (LAPC4: p<0.001, LNCaP: p<0.001, 22Rv1: p<0.001). Similarly, CAFs decreased darolutamide toxicity in LAPC4 and LNCaP cells (both p<0.001), while as expected 22Rv1 showed no response to darolutamide. Myofibroblasts had no significant effect on treatment efficacy. In the LumeNEXT system, CAFs again diminished darolutamide and docetaxel-induced cell death in LNCaP cells compared to conditions without CAFs (Docetaxel: p<0.001, Darolutamide: p<0.001). Our findings indicate that CAFs can attenuate the cytotoxic effects of ARPIs and docetaxel in 3D PCa TME models, highlighting their crucial role in treatment resistance. Ongoing research will further investigate the impact of patient-derived fibroblasts and the underlying molecular mechanisms through single-cell RNA sequencing. Nikolett Lupsa, Erika Heninger, Kate Vietor, Shannon R. Reese, Xavier T. Hazelberg, Jacob Popp, Aaron M. LeBeau, David J. Beebe, Sheena C. Kerr, Joshua M. Lang. : Modeling CAF-mediated therapy resistance in 3D prostate cancer systems using STACKs and LumeNEXT [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr A042.

Miniaturized biological assays using microfluidics have the potential to enhance assay sensitivity, reduce reagent consumption, and increase throughput. However, challenges to miniaturization include increased platform complexity and increased surface to volume ratios leading to risk of evaporation and analyte loss through surface binding. Exclusive Liquid Repellency (ELR) enables open microfluidic systems that minimize these challenges through an oil phase that protects small aqueous volumes from temperature fluctuation and evaporation while eliminating surface fouling that leads to sample loss. Here we report a novel microfluidic platform leveraging ELR and Exclusion-based Sample Preparation (ESP) for the miniaturization of CUT&Tag, a complex multistep biological assay. The resultant Lossless Altered Histone Modification Analysis System (LAHMAS) employs a PDMS-silane treated glass surface immersed in silicone oil to facilitate lossless liquid handling and prevent sample evaporation. The device design, compatible with standard laboratory equipment, allows effective processing of cell inputs as low as 100 cells with higher specificity than macroscale CUT&Tag facilitating accurate chromatin profiling of low input and rare cell samples.

Osteoclasts are specialized cells that degrade the bone matrix to create space for bone regeneration. During tumorigenesis, cancer cells metastasize to bone by disrupting bone's natural remodeling cycle. However, the mechanisms underlying critical bone-tumor interactions are poorly understood due to challenges in isolating osteoclasts from human bone. Thus, the conventional method to obtain osteoclasts for in vitro studies is via the differentiation of peripheral blood monocytes, which results in mixed cultures containing progenitor cells and osteoclasts of varying maturity and nuclearity. Presently, we hypothesized that the transcriptomic signatures of mature, multinucleated osteoclasts are distinct from osteoclasts with fewer nuclei. We established a live cell biomarker expression-based sorting protocol to allow purification of mature osteoclasts while maintaining viability and function. We observed that mature, multinucleated osteoclasts were transcriptomically distinct from those with fewer nuclei and that mature osteoclasts showed higher expression of genes that are associated with osteoclast fusion and function.

BACKGROUND:Culturing neuronal cells in vitro, especially at smaller scales with reduced media volumes, has been challenging due to the limited proliferation of mature neurons and the inherent high sensitivity of neuronal cells to environmental fluctuations. NEW METHOD:In this study, we report a neuronal cell culture method that leverages oil overlay and an autonomously regulated oxygen microenvironment (AROM), in which primary rat cortical cells and human neural progenitor cells (NPCs) were cultured in standard well plates with an oil overlay on top of the media layer. The oil overlay prevents evaporation and achieves in vivo-like oxygen concentrations without the use of glove boxes or hypoxic chambers. RESULTS:This oil overlay method achieved > 95 % yield of viable replicates after up to 30 days. Human NPCs cultured under the oil overlay for 15 days exhibited sustained viability without requiring media change. Additionally, oil overlays create a modulated oxygen microenvironment (i.e., AROM) that mimics in vivo conditions, capable of maintaining and restoring optimal oxygen concentrations after disturbances. COMPARISON WITH EXISTING METHOD:In contrast, existing method (no-oil controls) resulted in < 20 % yield, low viability for human NPCs (11 % versus 89 % with oil overlay), and oxygen concentrations that returns to ambient levels (21 % oxygen). CONCLUSION:Overall, these results support the oil overlay method as a robust small-scale neuronal cell culture system, offering improved stability and higher yield. The results also underscore the critical role of the oxygen microenvironment in supporting neuronal cell viability, maintenance, and growth.

Biofilms are three- dimensional structures containing one or more bacterial species embedded in extracellular polymeric substances. Although most biofilms are stationary, Flavobacterium johnsoniae forms a motile spherical biofilm called a zorb, which is propelled by its base cells and contains a polysaccharide core. Here, we report the formation of spatially organized, motile, multispecies biofilms, designated "co- zorbs," that are distinguished by a core-shell structure. F. johnsoniae forms zorbs whose cells collect other bacterial species and transport them to the zorb core, forming a co-zorb. Live imaging revealed that co-zorbs also form in zebrafish, thereby demonstrating a different type of bacterial movement in vivo. This finding opens different avenues for understanding community behaviors, the role of biofilms in bulk bacterial transport, and collective strategies for microbial success in various environments.

Persistent neutrophilic inflammation can lead to tissue damage and chronic inflammation, contributing to non-healing wounds. The resolution phase of neutrophilic inflammation is critical to preventing tissue damage. Animal models have provided insight into resolution of neutrophilic inflammation via efferocytosis and reverse migration (rM); however, species-specific differences and complexity of innate immune responses make translation to humans challenging. Thus, there is a need for in vitro systems that can elucidate mechanisms of resolution of human neutrophilic inflammation. Here, a human microphysiological system (MPS) is developed to mimic an inflammatory sterile injury (SI) microenvironment to study the role of macrophage-derived extracellular vesicles (M-EVs) in the resolution of inflammation via neutrophil rM. The MPS integrates a blood vessel mimic, injury site spheroid, human neutrophils, macrophages, and macrophage-derived EVs to investigate the role of M-EVs in neutrophil rM in vitro. The MPS enabled demonstration that EVs derived from macrophage subsets modulate migratory behavior in primary neutrophils differently in specific inflammatory microenvironments. A new mechanism is identified underlying neutrophil rM, where neutrophils exposed to M2-EV-derived-IL-8 migrate away from the SI site. Overall, the SI MPS system demonstrates a reverse migratory pattern in human primary neutrophils, advancing the study of the resolution of inflammation via M-EVs.

Oral transmission of parasites via environmentally resistant cyst stages in contaminated food or water is a common route of human infection, but there are no effective vaccines available for any enteric parasitic infection. Our knowledge of parasite cyst stage conversion and interaction with the intestinal tract is limited. Here, we investigate infection dynamics of Toxoplasma gondii cyst-stage in murine jejunum and human intestinal microphysiological systems. We focus on parasite ingress, replication, and conversion of the cyst stage to the rapidly replicating dissemination stage. In vivo bioluminescent imaging of mice fed cysts revealed spots of infection throughout the jejunum and ileum, which were selected for further analyses. Immunostaining showed parasite migration and replication predominantly in the stroma, with minimal replication in enterocytes. We recapitulated bradyzoite infection in human intestinal microphysiological systems and showed stage conversation and migration through collagen. This integrated approach elucidates complex host-parasite interactions, highlighting the value of microphysiological systems in advancing understanding and identifying potential therapeutics.

For patients with locally advanced, p16-negative head and neck squamous cell carcinoma (HNSCC), overall survival remains poor due to primary locoregional failure and distant metastasis following curative therapy. We aimed to understand how MAPKAPK2 (MK2) regulates HNSCC tumor cell migration and invasion, important first steps in cancer metastases. The TCGA database and HNSCC tissue microarrays were used to show that MK2 expression was associated with more advanced cancers and faster cancer recurrence rates. We observed that silencing of tumor MK2 in human cell lines (shRNA) caused a significant reduction in tumor cell migration-invasion in a complex HNSCC microphysiologic system used to recapitulate the tumor microenvironment. Murine cells (Ly2) with MK2 silenced (CRISPR-Cas9) also demonstrated reduced migration and invasion using 2D and 3D monoculture cell migration-invasions assays. Ly2 cells are orthotopic p16-negative murine metastatic cells that spontaneously metastasize, and we observed that MK2 inhibition via genetic (Cas9/CRISPR) or pharmacologic (PF-3644022) methods led to a significant reduction in the number of circulating tumor cells, fewer lymph node and lung metastases, and MK2 inhibited mice showed improved overall survival. Our findings suggest that HNSCC MK2 regulates tumor cell migration-invasion and may be a promising therapeutic target to reduce metastases.

Leukocytes forge paths through interstitial spaces by exerting forces to overcome confining mechanical pressures provided by surrounding cells. While such mechanical cues regulate leukocyte motility, engineering an in vitro system that models the deformable cellular environment encountered in vivo has been challenging. Here, microchannels are constructed with a liquid-liquid interface that exerts confining pressures similar to cells in tissues, and thus, is deformable by cell-generated forces. Consequently, the balance between migratory cell-generated and interfacial pressures determines the degree of confinement. Pioneer cells that first contact the interfacial barrier require greater deformation forces to forge a path for migration, and as a result migrate slower than trailing cells. Critically, resistive pressures are tunable by controlling the curvature of the liquid interface, which regulates motility. By granting cells autonomy in determining their confinement, and tuning environmental resistance, interfacial deformations match those of surrounding cells in vivo during interstitial neutrophil migration in a larval zebrafish model. It is discovered that neutrophils employ a bleb-based mechanism of force generation to deform a soft barrier exerting cell-scale confining pressures. In all, this work introduces a tunable in vitro material interface that replicates confining pressures applied by soft tissue environments.

Background: Head and neck cancer (HNC) treatment outcomes are highly variable among patients, and lack of actionable data on individual treatment responses limits effective clinical decision-making. Microphysiological systems (MPS) are advanced in vitro models that replicate the biological, physical, and functional properties of human tissues, offering a promising approach to testing treatment responses in vitro. Despite their potential, MPS adoption for evaluating standard clinical treatments—such as radiation and chemotherapy—has been hindered by the challenge of recreating the complexity of the tumor microenvironment. To address this, we developed an HNC MPS that mimics patient-specific tumor microenvironments using primary cells from patient tumors embedded in a 3D matrix with vascular components. This model was used to evaluate individualized responses to radiation and chemotherapy through cell-scale readouts, including DNA damage, proliferation, and migration. Methods: The HNC MPS comprises a microscale chamber containing two removable rods. A collagen-fibronectin matrix incorporating patient-derived tumor spheroids, fibroblasts, and immune cells was cast in the chamber. The matrix polymerized around the removable rods, which were subsequently extracted to create molded luminal structures. These lumens were lined with vascular and lymphatic endothelial cells to recreate a vascularized tumor microenvironment. An optimized media formulation supported the growth of all cell types within the MPS. The model was treated with radiation, cisplatin, or both to simulate the current standard of care. Cellular responses, including viability, proliferation, migration, and DNA damage, were assessed using immunofluorescence and cytokine secretion. A custom image processing pipeline analyzed multiple orthogonal readouts to reveal treatment responses at the cellular level with metrics to compare to actual patient outcomes under development. The model was further validated by comparing it with tumor tissue using single-cell RNA sequencing (scRNA-seq). Experiments were conducted with samples from multiple patients. Results: We successfully developed a patient-specific model of the HNC microenvironment using primary cells isolated from patient tumor tissue. This model captured key patient-specific features, such as angiogenesis and cell migration, and provided insights into treatment responses across different modalities. Cellular responses, such as proliferation and migration, were quantified, and scRNA-seq demonstrated close concordance between the MPS and patient tumor tissue. Our model presents a valuable functional assay for predicting patient-specific responses to therapy and validating biomarkers for treatment stratification. Conclusion: The HNC MPS is a robust in vitro platform that recapitulates patient-specific tumor microenvironments and treatment responses. It offers a promising approach for personalized cancer therapy, enabling functional assays to guide clinical decision-making and improve treatment outcomes for patients with HNC. Citation Format: Adeel Ahmed, Nathan W Hendrikse, Marcos A Lares, Fauzan Ahmed, Adam R Burr, Paul M Harari, David J Beebe, Sheena C Kerr. Patient-specific, organotypic head and neck cancer model for personalized medicine [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Functional and Genomic Precision Medicine in Cancer: Different Perspectives, Common Goals; 2025 Mar 11-13; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(5 Suppl):Abstract nr B023.

Bone is the most common site of prostate cancer metastasis, leading to significant morbidity, treatment resistance, and mortality. A major challenge in understanding treatment response is the complex, bone metastatic niche. Here, we report the first patient-specific microphysiological system (MPS) to incorporate six primary human stromal cell types found in the metastatic bone niche (mesenchymal stem cells, adipocytes, osteoblasts, osteoclasts, fibroblasts, and macrophages), alongside an endothelial microvessel, and prostate tumor epithelial spheroids in an optimized media that supports their viability and phenotype. We tested two standard of care drugs, darolutamide and docetaxel, in addition to sacituzumab govitecan (SG), currently in clinical trials for prostate cancer, demonstrating that the MPS accurately replicates androgen response sensitivity and captures stromal microenvironment-mediated resistance. This advanced MPS provides a robust platform for investigating the biological mechanisms of treatment response and for identification and testing of therapeutics to advance patient-specific MPS towards personalized clinical-decision making.

Abstract Bone metastases (BM) are the most common sites of metastases in prostate cancer, occurring in ~85% of patients. Overall survival of men with castrate resistant prostate cancer with BM is less than 24 months. Docetaxel is the most commonly used therapy for patients with prostate cancer, but the response rate is only 30-40% and median duration of response is less than 9 months. Non-tumor components of the Tumor Microenvironment (TME) have been proposed to mediate treatment resistance, but few pre-clinical models capture the complex physiology of the human bone TME. We report the development of a bone TME using a humanized microphysiologic system to address this need. The LumeNEXT platform is a microphysiologic system that allows the 3-dimensional (3D) reconstitution and integrated analysis of a bone-specific TME microenvironment with functional microvasculature that traverses the chip and can be used to mimic drug delivery in patients. Primary human osteoclasts (OC) were differentiated from patient peripheral blood monocytes. LNCaP prostate cancer spheroids were grown in hanging droplets. We performed co-culture of OCs with 3D LNCaP spheroids and evaluated the efficacy of docetaxel-induced tumor cell killing in the presence of bone cells. OCs and PC spheroids were seeded into LumeNEXT devices in a collagen-based matrix and treated with 20 nM Docetaxel or DMSO vehicle for 48 hours. Docetaxel response was assessed using confocal microscopy for single spheroid diameter and cytotoxicity was measured using fluorescent cell death markers. Docetaxel-mediated killing of tumor spheroids was found to be significantly attenuated in the presence of OCs. In LNCaP only conditions, average Docetaxel-mediated cell death was 68.82%, compared to 53.86% in the LNCaP and OC co-culture condition (p<0.0001). Furthermore, Docetaxel treatment resulted in reduced tumor spheroid diameter compared to DMSO control when prostate cancer spheroids were cultured alone (p=0.0118). Additionally, the presence of OCs attenuated Docetaxel-induced morphological changes. Our data suggest that osteoclasts play a significant role in mediating chemotherapy response in prostate cancer. Future studies will examine transcriptional changes in prostate cancer cells that may be associated with Docetaxel resistance in the bone microenvironment and identify mechanisms of BM microenvironment-mediated drug resistance. Bioinformatic analysis of patient sequencing datasets is ongoing to identify osteoclast-associated signatures of treatment resistance. Citation Format: Adeline B. Ding, Erika Heninger, Shannon R. Reese, Cristina Sanchez-de-Diego, Ravi C. Yada, Nan Sethakorn, Sheena C. Kerr, Xavier T. Hazelberg, Marina N. Sharifi, David J. Beebe, Joshua M. Lang. Osteoclasts mediate chemotherapy resistance in a fully humanized microphysiologic system of prostate cancer bone metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 635.