Biology of AR+ prostate cancer metastases and the role of the microenvironment

Division Of Basic Sciences - Nci

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Abstract

LuCaP 136 model establishment and initial characterization: Following intracardiac injection of single cells, bone metastasis can be visualized using bioluminescence imaging (BLI) by 3 weeks after inoculation, and tumors grow exponentially with mice requiring euthanasia between 6-7 weeks. A large percentage of mice are paralyzed due to the outgrowth of metastasis in vertebrae. Metastasis also can be found in liver, adrenal, and kidney, but the predominant burden is in bone. Castration results in a dramatic slowing in tumor burden accumulation, and importantly, a general shift to heterogeneous tumor phenotypes. Of particular interest, we determined that soft tissue liver metastases are enriched for the NE phenotype compared to bone following castration (see below), and similar to human mPC. Tumor phenotypes: Serial sections of tissues containing metastasis were stained for eight markers, including BrdU and pHH3 proliferation markers. One form of analysis co-registered sections using HALO quantitative image analysis software, where 70 micron bins from the image scans were aggregated to analyze co-expression of markers. Bins containing multidimensional marker quantifications were subsequently clustered by similarity and projected in 2D space as UMAPs. There is a dramatic phenotypic shift and gain in heterogeneity from the intact to late castration tumors, with relatively few marker positive cells at the early castration stage. Intact tumor cells lose AR and proliferation markers, while small clusters of NR3C1(GR) positive cells are either maintained or newly gained. Late castration metastases diffusely express SOX2, as expected, and in addition, we discovered CDKN1A (p21) is frequently observed in SOX2+ metastases. Of interest, ASCL1 and SYP are expressed predominantly in non-identical or non-adjacent cells within SOX2+ tumors, and proliferation is more associated with the ASCL1 subpopulation, suggesting SYP+ differentiation-associated quiescence. Molecular phenotypes: Molecular phenotyping of metastases are ongoing using bulk and single cell transcriptomic analyses. To gain sufficiently in-depth sequences for tumor cells, we determined that depletion of mouse cells from tumor-bearing bone marrows is the optimal approach. We have analyzed intact and early and late castrated samples as well as orthotopic tumors via RNAseq. Because of the quality and depth of sequencing in bulk samples, differential gene expression (DGE) in even small subpopulations are observable. DGE and pathway analysis revealed loss of AR signaling in castrated samples as expected. IFN and NFkB signaling was induced with castration, and neuro as well as gastrointestinal pathways became prominent in late vs early castration, demonstrating lineage plasticity. We have begun single cell (sc) RNAseq analyses in order to associate transcriptomic and pathway analyses with specific subpopulations of heterogeneous tumor cells. Of particular interest is the finding that the MYC pathway is overrepresented in most bone metastasis cellular phenotypic clusters as compared to matched adrenal clusters. Importantly, MYC pathway enrichment has been demonstrated previously in human and mouse bone metastases relative to primary tumors In the second model of interest, LuCaP23.1 have altered TP53 (TP53-/mut) and WNT (APC-/mut) pathways. LuCaP23.1 is a relatively slow growing model with bone metastasis detected two months after tumor cell inoculation in about 30% of mice. Tumor bearing mice can survive up to 6 months. Histologically, osteoblastic bone metastases occur mainly in vertebrae with extensive osteoid matrix surrounding marrow spaces that contain tumor cells. ARPC LuCaP23.1 tumors (CK8+CDH1+) maintain strong AR expression in the bone microenvironment. Because clonally-derived osteoblastic metastases have not been investigated previously, our initial goal is to determine the cross-communication between tumor cells and the osteoblast lineage cells in the bone marrow. LuCaP167 is a PDX-derived CRPC model that expresses AR-V7. Following cardiac injection, the model localizes mainly to the bone to form both clonal-separated osteoblastic and osteolytic metastases. A minority of tumors form in the adrenal glands. This model shows no sensitivity to castration, as anticipated. The model will serve as a good preclinical test for therapeutics directed to the AR-V7 variant.