Novel Dormancy Mechanism of Castration Resistance in Bone Metastatic Prostate Cancer Organoids.

Advanced prostate cancer (PCa) patients with bone metastases are treated with androgen pathway directed therapy (APDT). However, this treatment invariably fails and the cancer becomes castration resistant. To elucidate resistance mechanisms and to provide a more predictive pre-clinical research platform reflecting tumor heterogeneity, we established organoids from a patient-derived xenograft (PDX) model of bone metastatic prostate cancer, PCSD1. APDT-resistant PDX-derived organoids (PDOs) emerged when cultured without androgen or with the anti-androgen, enzalutamide. Transcriptomics revealed up-regulation of neurogenic and steroidogenic genes and down-regulation of DNA repair, cell cycle, circadian pathways and the severe acute respiratory syndrome (SARS)-CoV-2 host viral entry factors, ACE2 and TMPRSS2. Time course analysis of the cell cycle in live cells revealed that enzalutamide induced a gradual transition into a reversible dormant state as shown here for the first time at the single cell level in the context of multi-cellular, 3D living organoids using the Fucci2BL fluorescent live cell cycle tracker system. We show here a new mechanism of castration resistance in which enzalutamide induced dormancy and novel basal-luminal-like cells in bone metastatic prostate cancer organoids. These PDX organoids can be used to develop therapies targeting dormant APDT-resistant cells and host factors required for SARS-CoV-2 viral entry.

A PSMA-targeted bispecific antibody for prostate cancer driven by a small-molecule targeting ligand.

Despite the development of next-generation antiandrogens, metastatic castration-resistant prostate cancer (mCRPC) remains incurable. Here, we describe a unique semisynthetic bispecific antibody that uses site-specific unnatural amino acid conjugation to combine the potency of a T cell-recruiting anti-CD3 antibody with the specificity of an imaging ligand (DUPA) for prostate-specific membrane antigen. This format enabled optimization of structure and function to produce a candidate (CCW702) with specific, potent in vitro cytotoxicity and improved stability compared with a bispecific single-chain variable fragment format. In vivo, CCW702 eliminated C4-2 xenografts with as few as three weekly subcutaneous doses and prevented growth of PCSD1 patient-derived xenograft tumors in mice. In cynomolgus monkeys, CCW702 was well tolerated up to 34.1 mg/kg per dose, with near-complete subcutaneous bioavailability and a PK profile supporting testing of a weekly dosing regimen in patients. CCW702 is being evaluated in a first in-human clinical trial for men with mCRPC who had progressed on prior therapies (NCT04077021).

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient's bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Hypoxia-induced PIM kinase and laminin-activated integrin α6 mediate resistance to PI3K inhibitors in bone-metastatic CRPC.

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6ß1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6ß1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

Specific bone region localization of osteolytic versus osteoblastic lesions in a patient-derived xenograft model of bone metastatic prostate cancer.

OBJECTIVE: Bone metastasis occurs in up to 90% of men with advanced prostate cancer and leads to fractures, severe pain and therapy-resistance. Bone metastases induce a spectrum of types of bone lesions which can respond differently to therapy even within individual prostate cancer patients. Thus, the special environment of the bone makes the disease more complicated and incurable. A model in which bone lesions are reproducibly induced that mirrors the complexity seen in patients would be invaluable for pre-clinical testing of novel treatments. The microstructural changes in the femurs of mice implanted with PCSD1, a new patient-derived xenograft from a surgical prostate cancer bone metastasis specimen, were determined. METHODS: Quantitative micro-computed tomography (micro-CT) and histological analyses were performed to evaluate the effects of direct injection of PCSD1 cells or media alone (Control) into the right femurs of Rag2-/-γc-/- male mice. RESULTS: Bone lesions formed only in femurs of mice injected with PCSD1 cells. Bone volume (BV) was significantly decreased at the proximal and distal ends of the femurs (p < 0.01) whereas BV (p < 0.05) and bone shaft diameter (p < 0.01) were significantly increased along the femur shaft. CONCLUSION: PCSD1 cells reproducibly induced bone loss leading to osteolytic lesions at the ends of the femur, and, in contrast, induced aberrant bone formation leading to osteoblastic lesions along the femur shaft. Therefore, the interaction of PCSD1 cells with different bone region-specific microenvironments specified the type of bone lesion. Our approach can be used to determine if different bone regions support more therapy resistant tumor growth, thus, requiring novel treatments.

Tumor infiltrating B-cells are increased in prostate cancer tissue.

BACKGROUND: The presence of increased B-cell tumor infiltrating lymphocytes (TILs) was seen in mouse prostate cancer (PCa) but has not been fully documented in human PCa. We, therefore, investigated the density of infiltrating B cells within human PCa utilizing a quantitative computational method. METHODS: Archived radical prostatectomy specimens from 53 patients with known clinical outcome and D'Amico risk category were obtained and immunohistochemically (IHC) stained for the B cell marker, CD20. Slides were reviewed by a genitourinary pathologist who manually delineated the tumoral regions of PCa. Slides were digitally scanned and a computer algorithm quantified the area of CD20 stained B-cells as a measure of B cell density within the outlined regions of prostate cancer (intra-tumoral region), versus extra-tumoral prostate tissue. Correlations were analyzed between B-cell density and demographic and clinical variables, including D'Amico risk groups and disease recurrence. RESULTS: For the entire cohort, the mean intra-tumoral B cell density was higher (3.22 SE = 0.29) than in the extra-tumoral region of each prostatectomy section (2.24, SE = 0.19) (paired t test; P < 0.001). When analyzed according to D'Amico risk group, the intra-tumoral B cell infiltration in low risk (0.0377 vs. 0.0246; p = 0.151) and intermediate risk (0.0260 vs. 0.0214; p = 0.579) patient prostatectomy specimens did not show significantly more B-cells within the PCa tumor. However, patient specimens from the high-risk group (0.0301 vs. 0.0197; p < 0.001) and from those who eventually had PCa recurrence or progression (0.0343 vs. 0.0246; p = 0.019) did show significantly more intra-tumoral CD20+ B-cell staining. Extent of B-cell infiltration in the prostatectomy specimens did not correlate with any other clinical parameters. CONCLUSIONS: Our study shows that higher B-cell infiltration was present within the intra-tumoral PCa regions compared to the extra-tumoral benign prostate tissue regions in prostatectomy sections. For this study we developed a new method to measure B-cells using computer-assisted digitized image analysis. Accurate, consistent quantitation of B-cells in prostatectomy specimens is essential for future clinical trials evaluating the effect of B cell ablating antibodies. The interaction of B-cells and PCa may serve as the basis for new therapeutic targets.