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.

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.

Plasma thyroid hormone (TH) binding proteins (THBPs), including thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin (ALB), carry THs to extrathyroidal sites, where THs are unloaded locally and then taken up via membrane transporters into the tissue proper. The respective roles of THBPs in supplying THs for tissue uptake are not completely understood. To investigate this, we developed a spatial human physiologically based kinetic (PBK) model of THs, which produces several novel findings. (1) Contrary to postulations that TTR and/or ALB are the major local T4 contributors, the three THBPs may unload comparable amounts of T4 in Liver, a rapidly perfused organ; however, their contributions in slowly perfused tissues follow the order of abundances of T4TBG, T4TTR, and T4ALB. The T3 amounts unloaded from or loaded onto THBPs in a tissue acting as a T3 sink or source respectively follow the order of abundance of T3TBG, T3ALB, and T3TTR regardless of perfusion rate. (2) Any THBP alone is sufficient to maintain spatially uniform TH tissue distributions. (3) The TH amounts unloaded by each THBP species are spatially dependent and nonlinear in a tissue, with ALB being the dominant contributor near the arterial end but conceding to TBG near the venous end. (4) Spatial gradients of TH transporters and metabolic enzymes may modulate these contributions, producing spatially invariant or heterogeneous TH tissue concentrations depending on whether the blood-tissue TH exchange operates in near-equilibrium mode. In summary, our modeling provides novel insights into the differential roles of THBPs in local TH tissue distribution.

The development of microphysiological cell culture models (MPMs) that align with the throughput demands of drug and chemical testing are needed to help reduce animal testing, aide in the discovery of new drugs, and identify harmful chemical exposures. To address this need, we have developed a process for rapid prototyping MPM devices using computer numerical control (CNC) micromilling of commercially available microplates. Microchannels are cut out of the existing microplate structure and ports are drilled into the bottom of the wells to interface the wells. To test versatility and benchmark to another rapid-prototyping approach, we manufactured common microfluidic features into microplates using four different CNC mills as well as a 3D printer. Cell viability was assessed for polystyrene (PS) well plates and two 3D printed resins (MED610 and VeroClear) with the PS showing >2.5-fold increase in cell growth after three days. Machines were tested on their ability to create common device features including a traditional microfluidic device as well as a custom design incorporating complex geometries. Features were measured by confocal microscopy. We found that features including 1000μm ports, 800μm microchannels, 200μm phase-guides, and 500μm post arrays were machined and the range of CVs for features were 1.02-4.42, 1.32-3.50, 2.34-16.58, 6.25-16.40 respectively, while the 3D printed features exhibited maximal CVs of 20.98, 11.68, 23.60, and 10.01 for the same features. Predictably, more expensive machines generally showed higher accuracy and lower variation, but many features could be created accurately and precisely by inexpensive (<$3000) machines facilitating the broader use of this technology to create a user customizable platform to support the prototyping, development, and testing of human relevant models with broad applications across the life sciences. ### Competing Interest Statement BJ holds equity in Onexio Biosystems L.L.C JJ holds equity in Onexio Biosystems L.L.C

Abstract Patients with multifocal, locally advanced prostate cancer are at the highest risk of recurrence and death. Neoadjuvant therapies have limited response rates in this patient cohort and there remains a critical need to develop curative treatments. Prostate cancer (PC) shows marked heterogeneity, which is not limited to tumor cells but extends across immune and stromal compartments and has been linked to metastatic disease and therapeutic resistance. Cancer associated fibroblasts (CAFs) have been proposed to play a critical role in PC progression and invasion, but it is still unclear how CAFs interact with tumor cells. We evaluated CAFs from prostatectomy specimens as drivers of resistance to neoadjuvant androgen receptor signaling inhibitor (ARSi) and chemotherapy and assessed treatment response in co-culture systems in the context of CAF presence. We have utilized a novel radio-pathology tool that integrates PSMA PET/MRI scans to identify regions of interest that associate with heterogenous treatment response. MRI scans were utilized to print patient-specific 3D molds to micro dissect live tissue for subsequent cell sorting and molecular analyses. We have established a multi-parameter flow cytometry panel to characterize primary prostate fibroblasts from tumor foci and adjacent normal prostate tissue. In addition, co-culture assays were performed including immortalized CAF (hPrCSC-44) and MSC-derived fibroblast. To examine the tumor-promoting role of CAFs, fibroblasts were co-cultured with androgen-sensitive 3D tumor PC spheroids (LNCaP, C42B and LAPC4) in the integrated microfluidic STACKs platform that allows for the assessment of spatio-temporal interaction in a variety of culture microenvironments to model complex interactions. Cytotoxicity in 3D PC spheroids was assessed after Apalutamide, Darolutamide, or Docetaxel treatment by confocal microscopy. Integrated DNA, RNA, and radiology analysis of tumor biopsies have identified gene signatures associated with early biochemical recurrence. Most biopsies had low tumor purity, evidence of the cytotoxic impact of chemohormonal therapy while high tumor purity predicted high tumor expression. Poor clinical outcome was significantly associated with elevated stromal scores while epithelial and stromal enrichment were inversely correlated (rho= -0.80, p< 5 × 10−16). The presence of CAFs significantly decreased Docetaxel (C42B only vs C42B+CAF, 86.07% vs. 44.57%, respectively, p<0.001; LAPC4 only vs LAPC4+CAF, 70.98% vs 26.64%, respectively, p<0.001) and Darolutamide-induced (C42B only vs C42B+CAF, 52.59% vs 37.87%, respectively, p=0.0407). In conclusion, recurrent PC is linked to increased stromal content and the presence of CAFs supported tumor survival in response to ARSi and Docetaxel treatment. The molecular drivers of this phenomenon are currently being investigated. Citation Format: Nikolett Lupsa, Erika Heninger, Adeline Ding, Shannon R. Reese, Xavier T. Hazelberg, Aaron M. LeBeau, Brian P. Johnson, Peter P. Geiger, David J. Beebe, David A. Quigley, Joshua M. Lang. Contribution of cancer associated fibroblasts to treatment response and resistance in high-risk multifocal prostate cancer [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 298.

The purpose of this study is to analyze the angular variations within Cupid’s bow in patients with unoperated unilateral cleft lip (UCL). Angular features of Cupid’s bow were quantified in standardized presurgical photographs of children with UCL by 5 medical professionals specializing in craniofacial anomalies. The peaks and valley of Cupid’s bow were identified. A cleft side (CSA) and a noncleft side angle (NCSA) were delineated and measured by each expert. The data was pooled, and the angles were analyzed for symmetry. Cupid’s bow asymmetry was defined as a difference between NCSA and CSA ≥3°. Of the 37 patients studied, 29 were found to have asymmetry of Cupid’s bow with an average angle difference of 8.0° (95% CI: 6.6°–9.5°). Within this group,15 patients were found with acute asymmetry and 14 with obtuse asymmetry. Geometric analysis was performed on an example of a patient with acute asymmetry to demonstrate how correction of asymmetry can be considered during surgical repair. There is an asymmetry that exists in the Cupid’s bow of a significant number of patients with unoperated UCL. This finding not only adds to our understanding of UCL but may also have important implications when selecting the method/technique of surgical repair.

Chemical risk assessment still primarily relies on extrapolation of data from high-confidence in vivo studies. Emerging 21st Century Toxicology tools and approaches have potential to figure more prominently in chemical risk assessment, but many challenges in translating this research into assessments remain. One of these tools, the Adverse Outcome Pathway (AOP) Wiki provides a framework to map and evaluate adverse chemical dynamics, that is the biochemical and physiological effects that occur after chemical exposure. The AOP-guided targeted review of relevant literature, described here, shares similarities with a doctoral thesis or literature review but forces critical evaluation of each step in a pathway including those of central dogma. Additionally, it provides valuable translational regulatory relevance. Data gaps identified through this process can be targeted areas of study in the thesis itself to increase translational relevance. One of the challenges with this tool is that many AOPs are under- or undeveloped. To help fill this need, a concerted effort by subject matter experts to speed the development of AOPs supported under the Organization for Economic Cooperation and Development (OECD) framework would benefit this translational problem. As a case study, we present our experience developing AOP 460: Antagonism of Smoothened receptor leading to orofacial clefting (OECD AOP workplan project 1.101) as part of a graduate literature review. AOP development offers clear benefits to the regulatory and academic communities and increased dissemination of AOPs replete with the most current state of scientific knowledge will promote research translation and increased risk assessment capabilities.

Cellular stress granules arise in cells subjected to stress and promote cell survival. A cellular protein that localizes to stress granules is Z-DNA-binding protein 1 (ZBP1), which plays a major role in necroptosis, a programmed cell death pathway mediated by the kinase RIPK3. Here, we showed that the stress granule inducer arsenite activated RIPK3-dependent necroptosis. This pathway required ZBP1, which localized to arsenite-induced stress granules. RIPK3 localized to stress granules in the presence of ZBP1, leading to the formation of ZBP1-RIPK3 necrosomes, phosphorylation of the RIPK3 effector MLKL, and execution of necroptosis. Cells that did not form stress granules did not induce necroptosis in response to arsenite. Together, these results show that arsenite induces ZBP1mediated necroptosis in a manner dependent on stress granule formation.

The thyroid hormones (THs), thyroxine (T4) and triiodothyronine (T3), are under homeostatic control by the hypothalamic-pituitary-thyroid axis and plasma TH binding proteins (THBPs), including thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin (ALB). THBPs buffer free THs against transient perturbations and distribute THs to tissues. TH binding to THBPs can be perturbed by structurally similar endocrine-disrupting chemicals (EDCs), yet their impact on circulating THs and health risks are unclear. In the present study, we constructed a human physiologically based kinetic (PBK) model of THs and explored the potential effects of THBP-binding EDCs. The model describes the production, distribution, and metabolism of T4 and T3 in the Body Blood, Thyroid, Liver, and Rest-of-Body (RB) compartments, with explicit consideration of the reversible binding between plasma THs and THBPs. Rigorously parameterized based on literature data, the model recapitulates key quantitative TH kinetic characteristics, including free, THBP-bound, and total T4 and T3 concentrations, TH productions, distributions, metabolisms, clearance, and half-lives. Moreover, the model produces several novel findings. (1) The blood-tissue TH exchanges are fast and nearly at equilibrium especially for T4, providing intrinsic robustness against local metabolic perturbations. (2) Tissue influx is limiting for transient tissue uptake of THs when THBPs are present. (3) Continuous exposure to THBP-binding EDCs does not alter the steady-state levels of THs, while intermittent daily exposure to rapidly metabolized TBG-binding EDCs can cause much greater disruptions to plasma and tissue THs. In summary, the PBK model provides novel insights into TH kinetics and the homeostatic roles of THBPs against thyroid disrupting chemicals.

Intercellular signaling drives human development, but there is a paucity of in vitro models that recapitulate important tissue architecture while remaining operationally simple and scalable. As an example, formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine epithelial-mesenchymal signaling through multiple pathways including the Sonic Hedgehog (SHH), transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), and epidermal growth factor (EGF) pathways. We have developed a robust, throughput-compatible microphysiological system to model intercellular signaling including epithelial-mesenchymal interactions that is useful for studying both normal and abnormal orofacial development. We describe the construction and operation of an engineered microplate created using CNC micromilling of 96-well microtiter plates capable of containing up to 20 epithelial-mesenchymal microtissues. A dense three-dimensional mesenchyme is created by embedding cells (O9-1, 3T3) in a biomimetic hydrogel. An epithelial layer is then overlayed on the microtissue by loading cells in engineered microchannels that flank the microtissue. The result is an engineering epithelial-mesenchymal interface that is both on and perpendicular to the imaging plane making it suitable for high-content imaging and analysis. The resulting microtissues and device are compatible with diverse analytical techniques including fluorescent and luminescent cell health and enzymatic reporter assays, gene expression analyses, and protein staining. This tractable model and approach promise to shed light on critical processes in intercellular signaling events in orofacial development and beyond.

BACKGROUND:Prostatic cancers include a diverse microenvironment of tumor cells, cancer-associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard-of-care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components. METHODS:We first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints. RESULTS:We identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard-of-care histopathology and viable multi-modal TME analyses. No single method was found that preserved cellular sub-types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse-transcription polymerase chain reaction and RNA-seq while isolation using antibody-labeled paramagnetic particles facilitated functional coculture experiments. CONCLUSIONS:The identification of candidate cases and use of these techniques enable translational research and the development of molecular and functional assays to facilitate prostate TME study without compromising standard-of-care histopathological diagnosis. This allows bridging clinical histopathology and further interrogation of the prostate TME and promises to advance our understanding of tumor biology and unveil new predictive and prognostic markers of prostate cancer progression.

INTRODUCTION AND OBJECTIVE: PSMA PET allows accurate localization of tumour recurrence in patients with biochemical recurrence post-radical prostatectomy. Correlations between histopathological parameters and disease recurrence are established. We aim to qualitatively and quantitatively assess topographic concordance of histopathological factors with PET local recurrences. METHODS: Our cohort was selected from the 100 men who received a F-DCFPyL PET scan through IMPPORT trial, a prospective non-randomised study for men with a rising PSA (>0.2 ng/mL) post prostatectomy. Patients with local recurrences were included in our study. Histopathological parameters including ISUP Grade Group, location of tumour, extraprostatic extension (EPE) and positive margins were collated from prostatectomy reports. Pre-defined concordance between the location of histopathological lesions and local recurrences were classified in three dimensional planes. Individual prostate heights and tumour locations were used to define likely tumour distances from the prostatic membranous urethral junction, and correlated with the height of the local recurrence from the vesicourethral anastomosis for quantitative analysis. RESULTS: 24 patients were eligible, median age was 71 yrs, median PSA 0.37 ng/ml and median time between prostatectomy and PET was 2.6 years. 15 patients had recurrences within the vesicourethral anastomotic region and 9 within the lateral surgical margins where the seminal vesicle (SV) bed lies. 100% concordance in the left to right plane between tumour location and local recurrence was found, with 79% of these lesions concordant three dimensionally. 42% of patients had three dimensional concordance between the location of their EPE and their local recurrence, and 21% of positive surgical margins. 17 of the 24 patients had recurrent lesions within their tumour height range (Graph 1). Five of these were recurrences were reported as lateral surgical margin recurrences. CONCLUSIONS: Local recurrence is highly concordant with the position of the tumour within the prostate. Low total concordance with positive surgical margins, raises concern that a negative surgical margin may not be as protective against local recurrence as previously hypothesised.

74 Background: Understanding the lethal nature of high risk prostate cancer, there is a need for the development of multimodal therapies. Prior studies have confirmed a survival benefit with the addition of docetaxel to androgen deprivation therapy (ADT) in men with metastatic hormone-sensitive prostate cancer (HSPC). We conducted a Phase II trial enrolling men with very high risk localized, locally advanced or oligometastatic prostate cancer (PC) to examine resistance and response to neoadjuvant chemohormonal therapy. This analysis aims to identify the preoperative predictors of biochemical recurrence (BCR). Methods: UW17009 is an IRB-approved open-label, single-arm trial that recruited 26 men with newly diagnosed advanced PC. Patients received ADT and docetaxel for 3 months followed by prostatectomy. The primary endpoint was pathologic complete response rate. A secondary clinical objective was the rate of PSA recurrence 12 months after surgery. The pre-trial PSAs, age, cancer grade, stage, percent tumor involvement of the initial biopsy, metastatic disease on conventional and 18F-DCFPyL PSMA (DCFPyL) PET/CT and MRI imaging, completion of chemohormonal therapy and PSA nadirs following chemohormonal therapy were assessed in relationship to biochemical recurrence. One way ANOVA was used to evaluate differences among continuous values: age, PSA at diagnosis, percent tumor involvement, and PSA nadir after chemo ADT. Fisher’s exact tests were used to evaluate the differences among categorical variables: stage at diagnosis, positive bone scan, and positive PSMA PET. Results: 26 patients were enrolled and underwent neoadjuvant treatment, radical prostatectomy (RP) and lymph node dissection. The median age was 62 (IQR 58-66), mean PSA at diagnosis was 32.8 ng/dl and 88.4% had Gleason 9 cancer. At study initiation, 12/26 patients had metastatic disease detected by DCFPyL-based PSMA PET. Final pathology demonstrated 81%(21/26) had ≥ pT3 and 73%(19/26) patients had negative margins. Positive lymph nodes were found in 10/26(38.5%) patients on final pathology. At week 6 after surgery, 91%(24/26) had undetectable PSA. At a mean follow up of 12.1 months(5.2-21.4), the biochemical recurrence rate is 58%(15/26). Features associated with BCR include stage, % tumor involvement, and positive PSMA PET scan. All patients with positive margins and 9/10 patients with positive nodes at final pathology developed BCR at a mean follow up of 12 months. Conclusions: In this neoadjuvant cohort, stage T2c, elevated PSA, positive pre-operative PSMA PET/CT, and PSA nadir > 1 following chemohormonal therapy predict biochemical recurrence. Clinically, and in the short term, neoadjuvant chemohormonal therapy prior to definitive surgery for very high risk localized and/or oligometastatic PC generates local tumor control with a high rate of negative surgical margins.

Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.

36 Background: Interest has arisen in the use of prostate specific membrane antigen (PSMA) PET/CT imaging to detect prostate cancer at metastatic sites using different tracers. Here, we examined the ability of 18F-DCFPyL (DCFPyL) PSMA-based PET imaging to detect nodal disease in comparison to conventional imaging in a cohort of men with locally advanced or oligometastatic prostate cancer (PC). Methods: UW17009 is an IRB-approved open-label, single-arm trial that enrolled 26 patients with newly diagnosed advanced PC. Patients received androgen deprivation therapy and docetaxel for 3 months followed by radical prostatectomy (RP) and pelvic lymph node dissection (PLND). Exploratory interventions include PSMA PET/CT and MRI imaging as a method for determining treatment response and heterogeneity in primary PC and metastatic lesions performed before and after chemohormonal therapy. Prior to randomization, patients received DCFPyL PET/CT and PET/MR imaging as well as CTs and Bone Scans. A mean dose of 7.86 mCi DCFPyL was administered. Whole-body PET/CT images were acquired starting at approximately 60 minutes after radiotracer injection followed by dedicated pelvic PET/MR and whole-body PET/MR. PET imaging findings were compared to conventional dedicated CT imaging and were correlated to the results of final pathologic examination of each pelvic nodal dissection. Results: 26 patients underwent conventional and exploratory imaging with subsequent neoadjuvant treatment, RP and PLND. The mean diagnostic PSA was 32.1 ng/dl and 88.5% had Gleason 9 PCa. Using conventional imaging, pelvic nodal disease was identified in 6/26 patients. Pelvic lymph node uptake was identified in 12/26 patients using DCFPyL-based PSMA PET. Initial correlation of the pathologic specimens with pretreatment PSMA PET imaging revealed pelvic nodal metastatic PC in 10/12(83%) patients. On a per-lymph node packet basis (6 per patient), there were 156 evaluable regions, including 65 from patients with positive nodes. PSMA detected 14 packets that were positive for PC and 102 packets that were negative on imaging and final pathology. PC was missed in 5 packets. The mean tumor size in the missed nodes was 2.3 mm(range 1-4 mm). Calculated sensitivity was 73.7%(95% CI [48.8, 90.8]), 85.7 % specificity(95% CI[78.1, 91.4]), and 95.3 % negative predictive value(95% CI[90.5, 97.7]). Conclusions: In comparison to conventional imaging, in this cohort, DCFPyL PSMA-based PET imaging identified nodal positive disease at twice the rate and when evaluating on a per-packet basis, there was high negative predictive value. Ongoing analysis of post-chemohormonal therapy PET imaging may provide more information regarding tumor response in this cohort.

The prostate tumor microenvironment (TME) is strongly immunosuppressive; it is largely driven by alteration in cell phenotypes (i.e. tumor-associated macrophages and exhausted cytotoxic T cells) that result in pro-tumorigenic conditions and tumor growth. A greater understanding into how these altered immune cell phenotypes are developed and could potentially be reversed would provide important insights into improved treatment efficacy for prostate cancer. Here, we report a microfluidic model of the prostate TME that mimics prostate ducts across various stages of prostate cancer progression, with associated stroma and immune cells. Using this platform, we exposed immune cells to a benign prostate TME or a metastatic prostate TME and investigated their metabolism, gene and cytokine expression. Immune cells exposed to the metastatic TME showed metabolic differences with a higher redox ratio indicating a switch to a more glycolytic metabolic profile. These cells also increased expression of pro-tumor response cytokines that have been shown to increase cell migration and angiogenesis such as Interleukin-1 (IL-1) a and Granulocyte-macrophage colony-stimulating factor (GM-CSF). Lastly, we observed decreased TLR, STAT signaling and TRAIL expression, suggesting that phenotypes derived from exposure to the metastatic TME could have an impaired anti-tumor response. This platform could provide a valuable tool for studying immune cell phenotypes in in vitro tumor microenvironments.

1264 Background: There is a clinical unmet need to develop and properly assess multimodal therapeutic regimens for men with newly diagnosed high risk and oligometastatic prostate cancer (PCa) given the lethal nature of this disease. Results from prior studies support a combined multimodality treatment approach with neoadjuvant chemohormonal therapy followed by cytoreductive radical prostatectomy in patients with newly diagnosed high-risk primary PCa with oligometastatic disease. We evaluated the utility of prostate-specific membrane antigen (PSMA)-based 18F-DCFPyL (DCFPyL) PET imaging for treatment response assessment to neoadjuvant chemohormonal therapy in men with high risk PCa. Methods: An IRB-approved open-label, single-arm prospective Phase II trial is enrolling patients with newly diagnosed PCa at UW-Madison (ClinicalTrials.gov Identifier: NCT03358563). Patient eligibility included men with high risk PCa (extracapsular extension (cT3a) or seminal vesicle involvement (cT3b) or invasion of adjacent structures (cT4), serum PSA >20 ng/mL or Gleason score of 8 to 10 and/or regional lymph node) or oligometastatic disease (metastases beyond regional lymph nodes defined as less than four bone metastases by conventional imaging (CT, bone scan) but no visceral metastases). Patients underwent a baseline DCFPyL pelvic PET/magnetic resonance imaging (PET/MRI) (PET1) followed by neoadjuvant chemohormonal therapy with androgen deprivation therapy and docetaxel for three cycles (3 months). This was followed by a repeat pelvis PSMA PET/MRI (PET2) prior to prostatectomy. Patients were clinically followed for 1 year after prostatectomy or/unless until they meet criteria for prostate specific antigen (PSA) failure criteria. Results: Patient characteristics: A total of 27 patients have been enrolled on this study to date, with 8 patients demonstrating PSA recurrence within the first year, 9 completing their 1 year follow-up, and 10 patients still in their one year follow-up period. Baseline biopsy Gleason score ranged from 7 to 9 (2 - Gl 7, 1 - Gl 8, 24 - Gl 9) with Gleason pattern ranging from 3-5. PSA at baseline ranged from 2.7 to 336 ng/mL (median 22.3, mean 35.3). Percent PSA level decreased on chemohormonal therapy ranged from 91.64% to 100% (median 98.6% and mean 98.1%) and absolute decrease in PSA level ranged from 317.30 to 4.38 ng/mL (median -20.5, mean -38.8). PET standardized uptake value (SUV) change: Primary PCa DCFPyL PET SUVmax at baseline PET1 ranged from 7.4 to 86.6 (median 25.5, mean 28.2) and post-chemohormonal PET2 ranged from 3.5 to 51.9 (median 8.2, mean 14.1). Percent change in SUVmax from PET1 to PET2 ranged from -83.12% to +72.97% (median -61.0%, mean -49.1%), and absolute SUVmax change from PET1 to PET2 ranged from -34.7 to +5.4 (median -12.2, mean -14.1). PET SUV change correlated with PSA: Baseline PET1 and PET2 SUVmax was moderately correlated with baseline and post-chemohormonal PSA levels, respectively (correlation coefficient of 0.58 and 0.59, respectively). Absolute SUVmax change was only weakly correlated with absolute PSA change (correlation coefficient 0.23), and percent SUVmax change was not correlated with PSA percent change. PET SUV change and PSA recurrence: Two of 27 patients showed an increased PSMA SUVmax on PET2, and both subsequently developed early PSA recurrence within 1 year (126 and 286 days post prostatectomy). Ten patients had less than 50% decrease in PSMA SUVmax on PET2, of whom 6 (60%) developed early PSA recurrence within one year of follow-up. Conclusions: Preliminary analysis demonstrates DCFPyL PSMA PET/MR can assess response to neoadjuvant chemohormal therapy in men with high-risk primary PCa. PET SUVmax was correlated with PSA and early PSA recurrence. Further analyses to explore correlation of PSMA PET with histopathological response, clinical outcomes and tumor microenvironment studies are ongoing.

334 Background: Previous studies have shown that addition of docetaxel to androgen deprivation therapy (ADT) significantly improves progression-free survival (PFS) and overall survival (OS) in men with metastatic hormone-sensitive PC. Removal of the primary may also improve outcomes by reducing tumor self-seeding. We are conducting a phase II trial in men with PC to examine the feasibility of NAC, response using PSMA PET/MRI imaging and molecular mechanisms of resistance. Methods: This is an open-label, single-arm trial. Thirty patients with newly diagnosed very high risk localized, locally advanced or oligometastatic PC will receive ADT/docetaxel for three cycles before prostatectomy. The primary endpoint is rate of complete pathologic response. Key secondary objectives include PSA recurrence at month 12 after surgery. Exploratory objectives include tumor response and response heterogeneity in primary and metastatic tumors before and after treatment assessed by PSMA PET/MRI and evaluation of gene expression signatures in cancer cells, prostate stroma, bone marrow microenvironment and circulating tumor cells. Results: To date, 26 of 30 patients have enrolled and completed treatment. Mean age was 61 and mean PSA at time of diagnosis was 32.1 ng/dl. All patients had multi-focal prostate cancer with 23/26 patients with Gleason Grade Group 5. Metastatic disease by conventional imaging was identified in 6/26 patients (5 in lymph nodes [LN] and bone, 1 in LN only). Treatment was overall well tolerated. All patients had multi-focal primary prostate cancer detected on PSMA PET/MRI. All patients had a decline in PSMA PET SUVmax in at least one intraprostatic lesion. Two patients had an increase in SUVmax in at least one intraprostatic lesion that correlated with a resistant tumor focus on histopathology. Conclusions: NAC prior to surgery generates high rates of local tumor control with a heterogeneous response between foci. Primary resistance, identified by increasing PSMA PET SUVmax, is uncommon, however incomplete responses were observed in nearly all patients, suggesting that more cycles of treatment would improve response. PSMA PET/MRI can be used to monitor response and resistance in PC. Clinical trial information: NCT03358563.