Cells in the Polyaneuploid Cancer Cell State Are Prometastatic

Abstract

Our research aims to understand the adaptive-ergo potentially metastatic-responses of prostate cancer to changing microenvironments. Emerging evidence implicates a role of the polyaneuploid cancer cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study revealed that PACC presence in patient prostate tumors at the time of radical prostatectomy was predictive of future metastasis. To test for a causative relationship between PACC state biology and metastasis in prostate cancer, we leveraged a novel method designed for flow cytometric detection of circulating tumor cells (CTC) and disseminated tumor cells (DTC) from animal models. This approach provides both quantitative and qualitative information about the number and PACC status of recovered CTCs and DTCs. Specifically, we applied this approach to the analysis of subcutaneous, caudal artery, and intracardiac murine models. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. Furthermore, in vivo colonization assays proved that PACC populations can regain proliferative capacity at metastatic sites. Additional in vitro analyses revealed a PACC-specific partial epithelial-to-mesenchymal transition phenotype and a prometastatic secretory profile, together providing preliminary evidence of prometastatic mechanisms specific to the PACC state. Implications: Considering that many anticancer agents induce the PACC state, our data position the increased metastatic competency of PACC state cells as an important unforeseen ramification of neoadjuvant regimens, which may help explain clinical correlations between chemotherapy and metastatic progression.

Figure 1.

Time-matched, subcutaneous injection of PC3-GFP-Luc parental population versus PACC-enriched population. A, Light microscopy photos and flow cytometric ploidy analysis of injected cells per injection group. B, Tumor volume and tumor weight measurements per injection group at experimental endpoint. C, Representative H&E photos of primary tumors per injection group and quantification of normalized PACC proportion, determined by automated QuPath measurement of nuclear sizes. D, Enumeration of CTCs sourced from the blood of each animal and quantification of >4N CTCs versus ≤4N CTCs. E, Enumeration of DTCs sourced from the bone marrow of each animal and quantification of >4N DTCs versus ≤4N DTCs.

Figure 2.

Size-matched, subcutaneous injection of PC3-GFP-Luc parental population versus PACC-enriched population. A, Light microscopy photos and flow cytometric ploidy analysis of injected cells per injection group. B, Tumor volume and tumor weight measurements per injection group at experimental endpoint. Kaplan–Meier curve indicating when mice reached tumor size-based experimental endpoint. C, Representative H&E photos of primary tumors per injection group and quantification of normalized PACC proportion, determined by automated QuPath measurement of nuclear sizes. D, Enumeration of CTCs sourced from the blood of each animal and quantification of >4N CTCs versus ≤4N CTCs. E, Enumeration of DTCs sourced from the bone marrow of each animal and quantification of >4N DTCs versus ≤4N DTCs.

Figure 3.

Caudal artery injection of PC3-GFP-Luc parental population versus PACC-enriched population. A, Light microscopy photos and flow cytometric ploidy analysis of injected cells per injection group. B, Representative BLI images capturing the cellular distribution and signal intensity immediately following caudal artery injection and 72 hours following caudal artery injection. C, Enumeration of DTCs sourced from the bone marrow of each animal and quantification of >4N DTCs versus ≤4N DTCs. D, Enumeration of DTCs sourced from the lung tissue of each animal and quantification of >4N DTCs versus ≤4N DTCs.

Figure 4.

Subcutaneous injection of PC3-Luc parental population vs. size-filtered PACC population. A, Light microscopy photos and flow cytometric ploidy analysis of injected cells per injection group. B, Representative BLI images capturing the cellular distribution and signal intensity immediately following subcutaneous injection and 28 days following subcutaneous injection. C, BLI images of 4 mock-injected and 12 PACC-injected mice 82 days following subcutaneous injection; note the different scale for mouse 2 and mouse 5. Quantification of the status of injected cells across all 12 PACC-injected mice at experimental endpoint. D, Weekly BLI flux of all experimental mice over 84 days.

Figure 5.

Intracardiac injection of PC3-GFP-Luc parental population versus PACC-enriched population. A, Light microscopy photos and flow cytometric ploidy analysis of injected cells per injection group. B, Representative BLI images capturing the cellular distribution and signal intensity immediately following intracardiac injection and 42 days following intracardiac injection. C, BLI images of 4 mock-injected and 4/7 PACC-injected mice that showed their first evidence of colonization between 91 and 112 days post intracardiac injection. D, Weekly BLI flux of all experimental mice over 135 days. Quantification of the status of injected cells across all injected mice at experimental endpoint.

Figure 6.

PACCs have a pEMT phenotype. A, RNA expression of a panel of EMT markers by RT-qPCR in a PC3-Luc parental population vs. size-filtered PACC population, in which each biological replicate reported is an average of three technical replicates. B, Protein expression of a panel of EMT markers by Western blot in a PC3-Luc parental population versus size-filtered PACC population and respective quantification by densitometry. Note that the bands for CDH1, VIM, SNAI. and CLDN1 are sourced from the same blot and use the same actin band for protein loading normalization calculations during densitometry. C, Summary table of RNA and protein expression. D, Representative immunofluorescent images of PACCs identified as DTCs in the bone marrow of a patient with castrate-resistant metastatic prostate cancer, stained for DAPI, an epithelial -origin cocktail, and VIM. CNV analyses of the corresponding single cells.

Figure 7.

PACCs have a prometastatic secretory profile. A, Transwell assay comparing the differential motility of a PC3-Luc population exposed to parental-conditioned media vs. PACC-conditioned media, in which each reported biological replicate is an average of two technical replicates. B, Cytokine array comparing the relative abundance of 274 cytokines of interest in parental-conditioned media versus PACC-conditioned media. C, Transwell assay comparing the effect of addition of recombinant IL6 on the motility of a PC3-Luc population. D, Single-cell tracking comparing the effects of addition of recombinant IL6 and/or tocilizumab on the motility of a PC3-Luc population.