Glypican-1 as a target for fluorescence molecular imaging of bladder cancer.

OBJECTIVES: To investigate whether anti-glypican-1 antibody Miltuximab conjugated with near-infrared dye IRDye800CW can be used for in vivo fluorescence imaging of urothelial carcinoma. METHODS: The conjugate, Miltuximab-IRDye800CW, was produced and characterized by size exclusion chromatography and flow cytometry with glypican-1-expressing cells. Balb/c nude mice bearing subcutaneous urothelial carcinoma xenografts were intravenously injected with Miltuximab-IRDye800CW or control IgG-IRDye800CW and imaged daily by fluorescence imaging. After 10 days, tumors and major organs were collected for ex vivo study of the conjugate biodistribution, including its accumulation in the tumor. RESULTS: The intravenous injection of Miltuximab-IRDye800CW to tumor-bearing mice showed its specific accumulation in the tumors with the tumor-to-background ratio of 12.7 ± 2.4, which was significantly higher than that in the control group (4.6 ± 0.9, P < 0.005). The ex vivo imaging was consistent with the in vivo findings, with tumors from the mice injected with Miltuximab-IRDye800CW being significantly brighter than the organs or the control tumors. CONCLUSIONS: The highly specific accumulation and retention of Miltuximab-IRDye800CW in glypican-1-expressing tumors in vivo shows its high potential for fluorescence imaging of urothelial carcinoma and warrants its further investigation toward clinical translation.

Platelet PD-L1 suppresses anti-cancer immune cell activity in PD-L1 negative tumors.

Strategies that interfere with the binding of the receptor programmed cell death protein-1 (PD-1) to programmed death ligand-1 (PD-L1) have shown marked efficacy against many advanced cancers, including those that are negative for PD-L1. Precisely why patients with PD-L1 negative tumors respond to PD-1/PD-L1 checkpoint inhibition remains unclear. Here, we show that platelet-derived PD-L1 regulates the growth of PD-L1 negative tumors and that interference with platelet binding to PD-L1 negative cancer cells promotes T cell-induced cancer cytotoxicity. These results suggest that the successful outcomes of PD-L1 based therapies in patients with PD-L1 negative tumors may be explained, in part, by the presence of intra-tumoral platelets. Altogether, our findings demonstrate the impact of non-cancer/non-immune cell sources of PD-L1 in the tumor microenvironment in the promotion of cancer cell immune evasion. Our study also provides a compelling rationale for future testing of PD-L1 checkpoint inhibitor therapies in combination with antiplatelet agents, in patients with PD-L1 negative tumors.

Wnt Signaling Drives Prostate Cancer Bone Metastatic Tropism and Invasion.

Wnt signaling has been implicated as a driver of prostate cancer-related osteoblast differentiation, and previous studies have linked modifications in Wnt function with the induction of tumor metastasis. A unique aspect of prostate cancer bone metastases in mouse models is their relative predilection to the hindlimb (femur) compared to the forelimb (humerus). Comparative gene expression profiling was performed within the humerus and femur from non-tumor-bearing mice to evaluate differences in the microenvironments of these locations. This revealed the relative overexpression of the Wnt signaling inhibitors WIF1 and SOST in the humerus compared to the femur, with increased WNT5A expression in femur bone marrow, suggesting a coordinated upregulation of Wnt signals within the femur compared to the humerus. Conditioned medium (CM) from bone marrow stromal cells (HS-5 cells) was used to mimic the bone marrow microenvironment, which strongly promoted prostate cancer cell invasion (3.3-fold increase in PC3 cells, P < .05; 7-fold increase in LNCaP cells, P < .05). WNT5A shRNA knockdown within the CM-producing HS-5 cells significantly decreased PC3 (56%, P < .05) and LNCaP (60%, P < .05) cell invasion. Similarly, preincubation of CM with WIF1 significantly blocked LNCaP cell invasion (40%, P < .05). shRNA-mediated knockdown of the Wnt receptors FZD4 and FZD8 also strongly inhibited tumor cell invasion (60% inhibition shFZD4, P < .05; 63% shFZD8, P < .05). Furthermore, small molecule inhibition of JNK, which is an important component of the noncanonical Wnt signaling pathway, significantly inhibited CM-mediated tumor invasion. Overall, this study reveals a role for Wnt signaling as a driver of prostate cancer bone metastatic tropism and invasion.

Circulating Tumor Cell-Based Molecular Classifier for Predicting Resistance to Abiraterone and Enzalutamide in Metastatic Castration-Resistant Prostate Cancer.

While circulating tumor cell (CTC)-based detection of AR-V7 has been demonstrated to predict patient response to second-generation androgen receptor therapies, the rarity of AR-V7 expression in metastatic castrate-resistant prostate cancer (mCRPC) suggests that other drivers of resistance exist. We sought to use a multiplex gene expression platform to interrogate CTCs and identify potential markers of resistance to abiraterone and enzalutamide. 37 patients with mCRPC initiating treatment with enzalutamide (n = 16) or abiraterone (n = 21) were prospectively enrolled for CTC collection and gene expression analysis using a panel of 89 prostate cancer-related genes. Gene expression from CTCs was correlated with PSA response and radioclinical progression-free survival (PFS) using Kaplan-Meier and Cox regression analyses. Twenty patients (54%) had detectable CTCs. At a median follow-up of 11.3 months, increased expression of the following genes was significantly associated with shorter PSA PFS and radioclinical PFS: AR, AR-V7, PSA, PSCA, TSPAN8, NKX3.1, and WNT5B. Additionally, high SPINK1 expression was associated with increased PFS. A predictive model including all eight genes gave an area under the curve (AUC) of 0.84 for PSA PFS and 0.86 for radioclinical PFS. In comparison, the AR-V7 only model resulted in AUC values of 0.65 and 0.64.These data demonstrate that clinically relevant information regarding gene expression can be obtained from whole blood using a CTC-based approach. Multigene classifiers in this setting may allow for the development of noninvasive predictive biomarkers to guide clinical management.

Platelet-Synthesized Testosterone in Men with Prostate Cancer Induces Androgen Receptor Signaling.

Platelets have been long postulated to play a critical role in the pathogenesis of prostate cancer, although relatively little is known regarding the precise mechanisms involved. Androgen deprivation therapy (ADT) for prostate cancer eventually fails with relapse occurring in the form of castration-resistant prostate cancer (CRPC). CRPC tumors typically overexpress androgen receptor (AR), demonstrating continued dependence upon AR signaling. Platelets have been previously demonstrated to contain androgens, and we sought to explore the contribution of platelet-derived androgens in CRPC. In this study, we examined the role of platelet-derived androgens in vitro using platelets from men with CRPC, men with high-risk prostate cancer, and healthy male donors. A series of in vitro assays was performed to elucidate the impact of platelet-derived androgens on androgen-sensitive prostate tumor cells. By examining platelet-derived androgen effects on AR signaling in prostate tumor cells, we found that platelets, from men with CRPC and on ADT, strongly induce AR target genes and tumor cell proliferation. Moreover, we show a fully intact testosterone (T) biosynthetic pathway within platelets from its precursor cholesterol and demonstrate that platelets of CRPC patients with ADT resistance are able to generate T. Overall, our findings reveal an unknown capacity of platelets to synthesize T at functionally relevant levels in patients with lethal prostate cancer. Importantly, it suggests a novel paracrine mechanism of T production that may act to sustain CRPC state and potentiate therapeutic resistance.