Immunohistochemical analysis of differentiation in static and mixed prostate cancer spheroids

Abstract

Neoplastic cells self-assemble into multicellular spheroids that resemble micrometastases and avascular regions of larger tumors [1]. The applications of these tissue constructs include basic oncological research on invasion [2] and angiogenesis [3], and applied research on drug development [4] and delivery [5]. Culture conditions are critical to spheroid composition for these applications. For instance, mixing culture medium enhances collagen content of regenerated cartilage and calcium deposition in bone constructs for implantation [6,7]. These examples are illustrative of an existing paradigm that normal tissue becomes more differentiated upon mixing. During malignant transformation, however, differentiation mechanisms are disrupted [8]. The present study tests the validity of AbstractNeoplastic multicellular spheroids are in vitro models of solid tumors employed in drug testing and basic research. This study compares differentiation in static and mixed prostate cancer spheroids. Staining intensity of prostate specific antigen (PSA) was down-regulated upon mixing, from 0.21 ± 0.03 to 0.13 ± 0.03 in LNCaP multicellular spheroids, and from 0.13 ± 0.04 to 0.03 ± 0.02 in DU 145 multicellular spheroids. This was accompanied by 65% increase in the expression of cytokeratins 8 and 18 in DU 145 spheroids. PSA expression extended 60 µm within static spheroids and was disrupted in mixed culture. Diminished PSA expression and spatial organization suggests a more aggressive cancer. Higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation. Thus, the existing paradigm of differentiation established for normal tissue does not apply for our neoplastic spheroids. Cell dedifferentiation is attributed to improved interstitial transport and synthesis of extracellular matrix.

Predicting Aggregation Kinetics of DU 145 Prostate Cancer Cells in Liquid-Overlay Culture

“…Spatial profiles in viability and differentiation arise from mass transport effects and metabolic activity. For example, expression of prostate specific antigen is limited to the outer cell layers of prostate cancer spheroids (Song et al 2003a). Given the relationship between spheroid size and its composition, mathematical models that predict a spheroid size distribution may also be useful in selecting a priori the inoculation concentration that will produce spheroids with a desired composition.…”

“…This engineered tissue has application to drug development/delivery, design of patient-specific therapies, and basic cancer research (Chung 2003, O'Connor 1999. A feature of spheroids is the dependence of spheroid composition on their size (Song et al 2003a). Our research group has recently developed a novel mathematical model for the aggregation of attachment-dependent cells that has application to spheroid self-assembly of human prostate cancer cells (Enmon et al 2002).…”

Three-Dimensional Cell Cultures as an In Vitro Tool for Prostate Cancer Modeling and Drug Discovery

“…This effect may be the consequence of a PSA gene feedback repression by PSA protein accumulated in Matrigel-cultured cells, while PSA produced by PC3 cells in monolayers may be quickly secreted into the supernatant [ 44 ]. Interestingly, mixing culture medium significantly downregulated PSA expression and spatial organization in DU145 spheroids, leading to tumor cell dedifferentiation presumably caused by improved interstitial transport and synthesis of extracellular matrix [ 45 ]. Many experiments are ongoing to further investigate if common PCa cell lines cultivated with 3D methods can retain the clinical/diagnostic features of the original tumor.…”

Cancer research by means of tissue engineering – is there a rationale?

“…Furthermore, spatial organization of the stained cells suggested a more aggressive form of cancer. The authors speculated that a higher cytokeratin expression could result from either differentiation towards a luminal phenotype or activation of the Ras pathway during dedifferentiation [ 43 ]. These findings are of interest because they contradict the existing paradigm of differentiation established for normal tissue.…”

“…These findings are of interest because they contradict the existing paradigm of differentiation established for normal tissue. Cell dedifferentiation could be attributed to improved interstitial transport and synthesis of extracellular matrix in the spheroid TE tumour model [ 43 ]. These experiments show that TE techniques in tumour research can help elucidate the principal phenomena of prostate cancer cell development and could potentially provide in vitro platforms for rapid immunotherapy development [ 42 ].…”