Abstract

Introduction:

Prostate Cancer-3 (PC-3) cells, commonly used as a model for aggressive, androgen-independent prostate cancer, display numerous genetic alterations that contribute to advanced disease, including the loss of tumor suppressors and dysregulated inflammatory signaling. Recent evidence has highlighted the pleiotropic roles of lipocalin 2 (LCN2) in promoting tumor cell proliferation, adhesion, and stress resistance. This study aimed to investigate the functional and molecular effects of LCN2 depletion in PC-3 cells.

Methods:

We conducted a genetic analysis of both the parental PC-3 cell line and a newly created LCN2-deficient PC-3 clone #1 (PC-3 LCN2-KO#1), developed using CRISPR/Cas9 technology. Short tandem repeat (STR) analyses confirmed the authenticity and lineage of each cell line, while next-generation sequencing coupled with RT-qPCR validation was used to identify differentially expressed genes and any potential genomic changes resulting from the CRISPR/Cas9 editing process.

Results and Discussion:

Our analysis aligned with our previous findings showing that LCN2 is involved in inflammation, endoplasmic reticulum stress responses, and cytoskeletal organization. Previously we have shown that LCN2-deficient cells exhibited decreased invasiveness, disrupted F-actin dynamics, and increased sensitivity to stress-inducing conditions. Consistent with these observations, spectral karyotyping (SKY) and analysis of spontaneously occurring micronuclei revealed an elevated level of chromosomal aberrations in the LCN2-deficient cell line. These results emphasize the significance of LCN2 in driving prostate cancer aggressiveness and provide a foundation for exploring targeted interventions that disrupt LCN2-mediated pathways in advanced disease.

1 Introduction

Prostate Cancer-3 (PC-3) cells were established in 1979 by Kaighn and colleagues from a bone metastasis in a 62-year-old patient with advanced grade IV prostate adenocarcinoma (Kaighn et al., 1979). These cells were developed to study the characteristics of aggressive, androgen-independent prostate cancer. They do not depend on androgens for growth, making them a valuable model for late-stage disease and offering insights into hormone-refractory conditions (Kaighn et al., 1979; Rudzinski et al., 2020).

Since they were isolated from a metastatic lesion, PC-3 cells naturally have altered regulatory mechanisms that promote aggressive traits, such as rapid growth and migratory behavior. Specifically, these cells produce several cytokines and growth factors that can potentially stimulate tumor innervation (Voss et al., 2010). Consequently, they have become essential in prostate cancer research, frequently used in drug screening, co-culture experiments, and in vivo models to explore therapeutic strategies for managing bone metastases. The cells are also utilized in xenograft models, with the ability to induce lymph node metastases (Wu et al., 2013).

Notably, PC-3 cells lack androgen receptors, which explains their unresponsiveness to androgen-deprivation therapies. However, other studies have shown the existence of a subline of PC-3 cells that produce measurable androgen receptor mRNA and traces of AR protein (Buchanan et al., 2004). They also contain genetic alterations like the loss of the tumor suppressor PTEN and deletions, as well as abnormalities in TP53 (Fraser et al., 2012; Chappell et al., 2012; Seim et al., 2017). Genome-wide analyses indicate that inflammation- and proliferation-related pathways are upregulated in these cells, further emphasizing their aggressive phenotype compared to other human prostate cancer cell lines (Dozmorov et al., 2009). Altogether, these features make PC-3 cells an indispensable tool for understanding the molecular basis of advanced prostate cancer and for guiding the development of innovative therapeutic strategies. Although they have been extensively studied, ongoing research continues to refine our understanding of their biology, highlighting the importance of verifying specific experimental details with the original literature.

Another recent significant application of the PC-3 cell line involves generating LCN2-deficient variants to study how this secreted protein influences prostate cancer behavior (Schröder et al., 2022). LCN2, also known as neutrophil gelatinase-associated lipocalin (NGAL), is a 25-kDa transporter protein belonging to a large family of small extracellular proteins involved in various cellular processes, including innate immunity, cellular adhesion, inflammatory signaling, and tumor progression (Asimakopoulou et al., 2016; Schröder et al., 2023a; Chandrasekaran et al., 2024). In a previous study, CRISPR/Cas9-mediated knockout and siRNA-based suppression of LCN2 in PC-3 cells were used to understand how LCN2 deficiency impacts metastatic properties (Schröder et al., 2022). Remarkably, LCN2-deficient PC-3 cells showed reduced proliferation and cell adhesion, disrupted F-actin organization, decreased expression of pro-inflammatory cytokines, and increased sensitivity to endoplasmic reticulum stress. Reduced IL-1β expression, along with decreased invasiveness and compromised stress response, further underscored the significance of LCN2 in advanced prostate cancer. These findings collectively demonstrate the usefulness of PC-3-based LCN2-knockout models in dissecting the molecular pathways involved in prostate cancer progression and validating LCN2 as a promising therapeutic target in aggressive disease stages (Schröder et al., 2022). Furthermore, disrupting LCN2 in these cells led to decreased activation of the JAK/STAT pathway and reduced expression of interferon-stimulated genes (Barer et al., 2023). All these findings suggest that comparing parental PC-3 cells with LCN2-deficient subclones derived from them is a valuable tool for investigating aspects of LCN2 function in prostate cancer.

In this paper, we will genetically characterize one of our newly generated PC-3 clones, designated as clone #1, and compare its mRNA and protein expression profile to that of the parental PC-3 cell line. Additionally, we conducted next-generation sequencing (NGS) on clone #1 and the parental PC-3 cell line to systematically evaluate gene expression changes in the absence of LCN2. This approach enabled us to identify molecular pathways that are differentially regulated when LCN2 is depleted, providing more insights into the role of lipocalins in prostate cancer progression. Furthermore, SKY analysis was used to compare the genome wide structural organization of clone #1 with that of the parental PC-3 cell line.

2 Materials and methods2.1 Literature search

A comprehensive review of relevant literature was conducted to identify studies using the PC-3 prostate cancer cell line, focusing on its genetic alterations, protein expression, and functional assays. Databases searched included PubMed, Web of Science, and Scopus. The search strategy involved combining the terms “PC-3,” “prostate cancer,” and “cell line,” along with “genetic,” “expression,” or “knockout.” Priority was given to articles published in English, and reference lists of key studies were examined to locate additional relevant publications. The selected papers informed both the experimental design and the interpretation of our results. Additionally, we explored the FDI Lab: SciCrunch Infrastructure database for references to PC-3 (SciCrunch).

2.2 Cell culture

PC-3 cells (CRL-1435; RRID:CVCL_0035) were obtained from the German branch (LGC Standards GmbH, Wesel, Germany) of the American Type Culture Collection (ATCC). Both, the parental PC-3 cells and the previously established clone #1 of PC-3 lacking LCN2 (referred to as PC-3 LCN2-KO#1) (Schröder et al., 2022) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, #D6171, Sigma-Aldrich, Merck, Taufkirchen, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS, #F7524, Sigma-Aldrich), 2 mM L-glutamine (#G7513, Sigma-Aldrich), and 1× penicillin/streptomycin (DE17-602E, Lonza, Cologne, Germany). The cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2 and split when they reached 70%–80% confluency. For subculturing, the cells were rinsed with phosphate-buffered saline (PBS) and detached using Accutase solution (#A6964-100ML, Sigma-Aldrich).

2.3 Light microscopy, FACS analysis, and phalloidin stain

Live-cell morphology was observed using a Leica DM IL LED microscope equipped with a Leica EC3 camera and the Leica Application Suite (LAS) software (version 3.4.0), all from Leica (Wetzlar, Germany), with phase-contrast, bright field, or fluorescent illumination. Images were captured using an attached digital camera with bright field or fluorescent illumination. Bright-field images were used to assess cell density, confluency, and any morphological changes indicating contamination or differentiation. For FACS analysis, both cell lines were grown in 10 cm plates for 2 days, until they reached approximately 80% confluency. Cells were then detached using Accutase® solution, collected by adding medium, and centrifuged at 300 rpm for 8 min at 4 °C. After discarding the supernatant, the resulting pellets were gently suspended in 1 mL of FACS buffer (10 mM HEPES, pH 7.3, 0.06% bovine serum albumin, and 0.3 mM EDTA) prepared in Hank’s balanced salt solution without phenol red, calcium and magnesium (#88284, Thermo Fisher Scientific). The samples were placed on ice, filtered through a 40 µm nylon mesh into dedicated FACS tubes, and briefly re-suspended before analysis. Flow cytometry was performed on a BD FACSAria II SORP, with BD FACSDiva™ 6.0 software used to process 10,000 events per sample. A forward scatter (FSC) threshold of 5,000 was set to exclude debris, and doublet discrimination was applied to the side scatter (SSC) and FSC channels. The proportion of GFP-positive cells was then recorded. Phalloidin staining of PC-3 cells and their derivatives was performed using a protocol that we have described before (Schröder et al., 2023b) and images were taken with a Nikon Eclipse E80i fluorescence microscope (Nikon Imaging Japan Inc., Shinagawa-ku, Tokyo, Japan).

2.4 Short tandem repeat profiling

To verify the authenticity of both parental PC-3 and clone #1 cells, short tandem repeat (STR) profiling was conducted at IDEXX Laboratories in Kornwestheim, Germany. The CellCheck™ Human 16 STR Profile and Interspecies Contamination Test were used for this purpose. The resulting STR profiles were then compared using the online Cellosaurus STR similarity search tool (CLASTR, version 1.4.4) (Cellosaurus CLASTR, 2025) with the following search parameters: Scoring: Tanabe; Modes: Non-empty markers; Filters: Score Filter: 60%, Min Markers: 8; Max Results: 200. In the Tanabe scoring system, also known as Sørensen-Dice coefficient, 15–17 polymorphic STR markers (typically 2-6 base pairs) are used to generate a profile that verifies cell line identity, ancestry, and sex (Amelogenin) (Tanabe et al., 1999).

2.5 Next-generation mRNA sequencing and data analysis

Total RNA from both parental PC-3 cells and clone #1 cells, isolated from cells grown to 70% confluence, underwent next-generation sequencing (NGS) to identify differentially expressed genes influenced by LCN2 deficiency. The RNA samples were initially assessed for concentration, purity, and integrity using UV spectrophotometry and an Agilent 4200 TapeStation (Agilent Technologies Inc., Waldbronn, Germany). After removal of ribosomal RNA, the remaining mRNA fraction was converted into a sequencing library using the NEBNext® Multiplex Oligos for Illumina® Index Primers Set 1 (#E7335S, New England Biolabs, Frankfurt am Main, Germany). These libraries were then sequenced on an Illumina instrument using 300-cycle MiSeq Reagent kit V2 cartridges (Illumina Inc., San Diego, CA, USA). All cDNA library preparations and sequencing procedures were conducted at the IZKF Genomic Facility, University Hospital Aachen. The resulting sequencing runs were demultiplexed and converted to FASTQ format using Illumina bcl2fastq conversion software (version 2.20), and these FASTQ files were utilized for subsequent downstream analyses. Raw sequencing reads were assessed for quality using FastQC (v0.11.8) (Babraham). Following the evaluation, adapters and bases with Phred scores below 20 were eliminated. Any reads shorter than 35 bp were discarded using cutadapt (v1.9.1) (Martin, 2011), and reads were aligned to the reference genome using STAR (v2.7.11a) (Dobin et al., 2013). Gene abundances were then estimated using featureCounts (v2.0.6) and the pseudoalignment-based methods kallisto (v0.48.0) (Bray et al., 2016) and salmon (v.1.10.2) (Patro et al., 2017), followed by median-of-ratios normalization (Love and Huber, 2014). For annotation purposes, the Human GRCh38.p14 reference transcriptome (Ensembl) was employed. To generate a candidate list of genes with potentially changed expression upon LCN2 depletion, which was later verified by RT-qPCR, we filtered out lowly expressed genes and only retained genes with log2 fold-changes ≥2 as estimated by featureCounts, kallisto, and salmon.

2.6 RNA extraction and real-time quantitative PCR

For targeted validation of selected differentially expressed genes, total RNA was extracted using the PureLink RNA Mini kit (#12183018A, Thermo Fisher Scientific Inc., Life Technologies GmbH, Darmstadt, Germany) following the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). First-strand cDNA was synthesized from 1 µg of total RNA using Superscript II Reverse Transcriptase (#18064–014, Thermo Fisher Scientific) with random hexamer primers (#C118A, Thermo Fisher Scientific). The resulting cDNA was diluted in RNase-free water and stored at −20 °C. For quantitative real-time PCR (RT-qPCR), 5 µL of diluted cDNA was combined with sequence-specific primers in a 25 µL reaction containing SYBR-Green™ qPCR SuperMix (#56465, Thermo Fisher Scientific). The thermal cycling protocol included an initial 10-min denaturation step at 95 °C, followed by 40 cycles of 15 s at 90 °C and 1 min at 60 °C. RT-qPCR measurements were performed in technical duplicates for each biological sample. The experiment was independently repeated three times, resulting in biological triplicates (n = 3). Expression levels were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative mRNA abundance was calculated using the 2−ΔΔCT method (Schmittgen and Livak, 2008). All primers used in the study are listed in Supplementary Table S1.

2.7 Western blot analysis

Protein expression levels were determined using Western blot analysis. Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer, which consisted of 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2% (w/v) NP-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, and the cOmplete™ protease inhibitor cocktail (#CO-RO, Merck, Darmstadt, Germany). Protein concentrations were measured using the DC Protein Assay (Bio-Rad Laboratories GmbH, Feldkirchen, Germany). Equal amounts of protein (40 µg) were separated by 10% SDS-PAGE under reducing conditions and transferred to 0.45 µm nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). Ponceau S stain was used to confirm successful protein transfer. The membranes were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (50 mM Tris, pH 7.6, 150 mM NaCl) containing 0.1% Tween 20. Subsequently, membranes were incubated overnight at 4 °C with primary antibodies targeting specific proteins, followed by horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. β-Actin was used as a loading control. Signals were detected using the Supersignal™ West Dura Extended Duration Substrate (#34076, ThermoFisher Scientific) and captured with an iBright™ 1500 Imaging System (Invitrogen, Thermo Fisher Scientific). The antibodies utilized in our study are listed in Supplementary Table S2.

2.8 Karyotype analysis and spectral karyotype analysis

Standard karyotype and SKY analyses were conducted on metaphase spreads from parental PC-3 and clone #1 cells to evaluate chromosomal composition. Both cell lines were cultured in T25 flasks at 37 °C until reaching semi-confluence. Subsequently, the cells were treated with colcemid, detached using mild trypsin-EDTA, and collected by centrifugation. After a 30-min hypotonic treatment with 0.56% KCl at 37 °C, the cells were fixed in methanol-acetic acid (3:1) solution. Air-dried chromosome spreads were prepared, and chromosome number and morphology were assessed using conventional Giemsa staining.

Chromosome rearrangements in both cell lines were examined using a commercial Human SKY probe from Applied Spectral Imaging in Carlsbad, CA. Slides aged 2–3 days were used for hybridization. Denaturation, in situ hybridization, and chromosomal counterstaining were carried out according to the manufacturer’s protocol. Images were captured using a Zeiss microscope AxioImager A1 (Jena, Germany). Spectral classification and pseudo-coloring of chromosomes were performed using HiSky 6.0 software (Applied Spectral Imaging, Carlsbad, CA, USA). At least 20 metaphases were analyzed by SKY for each cell line.

To validate the chromosomal rearrangements detected by SKY analysis, fluorescence in situ hybridization (FISH) was performed on metaphase spreads from both cell lines using commercially available chromosome-specific paints (Kreatech, Leica Biosystems, Amsterdam, Netherlands), following established standard protocols.

2.9 Data analysis and statistical analysis

Statistical analysis was performed with GraphPad Prism v.8.0 (GraphPad Software, Inc., La Jolla, CA). All data in this study are shown as mean ± standard deviation (SD). Statistical significance between groups was assumed in ANOVA test when probability values were below 0.05 (p < 0.05). The normality of data distribution was assessed using the Shapiro-Wilk test. For data that was normally distributed, statistical significance was evaluated using ordinary one-way ANOVA. For data that did not meet the assumptions of normality, the non-parametric Kruskal–Wallis test was applied. Significant differences are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

3 Results3.1 Usage of PC-3 cells in biomedical research

PC-3 cells are one of the most commonly used cell lines in prostate cancer research due to their androgen-independent status and aggressive metastatic characteristics. They have been cited in the Resource Identification Initiative (RRID) database over 1,000 times, indicating their widespread adoption in biomedical science. A PubMed search using the search terms “PC-3 or PC3” for the time period 1979 to 2025 yielded 17,627 entries (search conducted on 21 October 2025). These entries include studies from various disciplines, with the majority focusing on tumor progression, screening new drugs, and investigating the mechanistic pathways involved in advanced prostate cancer. Similarly, SciCrunch, which provides a curated database of reagents, tools, and materials, has reported a consistent increase in the use of PC-3 in recent years (Supplementary Figure S1).

3.2 Short tandem repeat profiling of parental PC-3 and PC-3 LCN2-KO clone #1

Short tandem repeat (STR) profiling was conducted to verify the genetic identity and authenticity of both the parental PC-3 cells and the derived clone #1. Analysis of 15 standard STR loci, along with the amelogenin sex marker, showed no discrepancies between the parental PC-3 cell line and the information provided by ATCC, or known references for PC-3 (Masters et al., 2001; van Bokhoven et al., 2003; Lorenzi et al., 2009; Fang et al., 2011; Yu et al., 2015) (Supplementary Table S3; Figure 1). However, clone 1, derived from PC-3 using CRISPR/Cas9 technology, exhibited losses of one STR variant each at variant sites D7S820 and TH01. The parental cell line displayed two signals indicating 8 and 11 repeats at variant site D7S820, and two signals indicating 6 and 7 repeats at variant site TH01, while clone 1 showed only a single signal at these sites. A similar loss at D7S820 was reported in PC-3 cells originating from the National Cell Bank of Iran (Azari et al., 2007). Nevertheless, the genetically modified cell line PC-3 LCN2-KO matched in 14 of the 16 tested markers. Based on the consensus standard, an identity match of 87.5% falls within the range of 80%–100%, confirming the similarity between the parental PC-3 cells and the PC-3 LCN2-KO clone (Korch et al., 2021). However, the absence of variant signals at D7S820 and TH01 may suggest genuine genomic changes in the cell population, such as partial deletions or clonal drift that eliminated these specific alleles.

Short tandem repeat (STR) profiling. Electropherograms for parental PC-3 (A) and CRISPR/Cas9-derived clone #1 (B) are shown. Each peak represents a specific STR locus, including the amelogenin sex marker. All loci matched the known PC-3 reference profile except for the loss of the two-allelic pattern at variant sites D7S820 (8 and 11 repeats in PC-3; 8 repeats in PC-3 LCN2-KO clone #1) and TH01 (6 and 7 in PC-3; 7 in PC-3 LCN2-KO clone #1). This confirms the authenticity and close genetic relationship of clone #1 to the parental cell line.

3.3 Microscopic, FACS analysis, and phalloidin stain

Microscopic examination and FACS analysis were conducted to evaluate cellular morphology and verify GFP fluorescence in CRISPR/Cas9-generated PC-3 LCN2-KO clone #1. Under bright-field illumination, the PC-3 cells lacking LCN2, including PC-3 LCN2-KO clone #1 and two other clones, appeared slightly rounded compared to the more elongated morphology of the parental PC-3 cells (Figure 2). To further assess cytoskeletal organization associated with this altered morphology in clones lacking LCN2, we stained filamentous actin (F-actin) using fluorescently labeled phalloidin (Supplementary Figure S1), again demonstrating the altered morphology compared to parental PC-3 cells. This observation confirms our previous finding that the loss of LCN2 may influence cytoskeletal organization and/or adhesion properties, thereby affecting overall cell shape (Schröder et al., 2022). Moreover, FACS analysis showed a clear GFP signal, originating from the CRISPR/Cas9 clone used to disrupt the LCN2 gene locus (Schröder et al., 2022), was detected exclusively in the three clones and not in parental PC-3 cells lacking this construct.

Light microscopic appearance and FACS analysis of PC-3 cells and PC-3 cells lacking LCN2. (A) Representative bright-field images show the cellular morphology in LCN2-deficient PC-3 cells compared to the original PC-3 line. (B,C) Flow cytometry was used to evaluate GFP expression from the CRISPR/Cas9 construct. A representative mixture of LCN2-KO cells (C) display a clearly visible GFP-positive population, while the original parental PC-3 cells (B) do not exhibit any GFP expression.

3.4 Karyotype analysis and spectral karyotype analysis

Cytogenetic analysis of Giemsa-stained metaphases from PC-3 and PC-3 LCN2-KO cells consistently showed a hyperdiploid karyotype, with no diploid cells identified. In approximately 90% of the analyzed metaphases, chromosome numbers ranged between 57 and 61, with a small proportion displaying tetraploidy. Due to the complexity of structural rearrangements that could not be accurately resolved by conventional GTG banding, spectral karyotyping (SKY) was subsequently used.

SKY analysis allowed for the simultaneous identification of all rearranged chromosomes, providing an overview of the chromosome segments involved in rearrangements across the analyzed metaphases. The large chromosomes HSA1, HSA2, HSA3, HSA4 and HSA5 were most frequently affected in both PC-3 and the derived PC-3 LCN2-KO cells, often present in multiple copies and exhibiting multiple translocations. Additionally, chromosomes HSA10, HSA11, HSA12, HSA14, HSA15, HSA17, and X also displayed rearrangements. Specific translocations in both karyotypes can be seen in Figures 3, 4 as well as the accompanying Supplementary Table S4.

Spectral karyotyping of a human PC-3 metaphase. An RGB image after hybridization with the SKY probe cocktail, containing chromosome-specific fluorescent probes in distinct colors (A) is shown alongside the spectrally classified pseudo-colored image (B) the corresponding inverted DAPI-stained image (C) and the karyotype. The karyotype below (D) presents spectrally classified pseudo-colored chromosomes (right), their inverted DAPI-stained counterparts (left), and the RGB images (middle). Chromosome segments involved in the rearrangements are labeled with their corresponding numeric and letter designations. The rearranged chromosomes are classified according to the presence of the largest segment from the respective chromosomes. Based on SKY in the majority of metaphases, the PC-3 karyotype can be designated as: <57-61>,XX,-Y,+1,der(1)t(1;8;10;12),+2,der(2)t(2;8)x2,der(2)t(2;15;17),+3,der(3)t(1;3;10)x2,der(3)t(3;10),+4,der(4)t(4;6),der(4)t(4;10)x2,der(4)t(4;12),+5,der(5)t(5;10;11;19),der(5)t(5;15;19),+7,der(7)t(7;18),-10,der(10)t(1;10;17),+11,der(11)t(7;11),+12,der(12)t(3;8;12;15;17),der(12)t(12;20),+14,der(14)t(14;16),-15,der(15)t(5;15;20),der(17)t(13;17),+20,+21,der(X)t(2;X)x2. (+) denotes the gain or presence of extra copies of a specific chromosome, whereas (−) denotes the loss or deletion of a specific chromosome.

Spectral karyotyping of a human PC-3 LCN2-KO metaphase. An RGB image after hybridization with the SKY probe cocktail, containing chromosome-specific fluorescent probes in distinct colors (A) is shown alongside the spectrally classified pseudo-colored image (B) the corresponding inverted DAPI-stained image (C) and the karyotype. The karyotype below (D) presents spectrally classified pseudo-colored chromosomes (right), their inverted DAPI-stained counterparts (left), and the RGB images (middle). Chromosome segments involved in the rearrangements are labeled with their corresponding numeric and letter designations. Translocated chromosomes were classified according to the chromosome from which the larger segment originated. Based on SKY in the majority of metaphases, the PC-3 LCN2-KO karyotype can be described as:<57-60>,XX,+Y, +1,der(1)t(1;8;10;12),der(1)t(1;18),+2,der(2)t(2;8)x2,der(2)t(2;15;17), +3,der(3)t(1;3;10),der(3)t(1;3;10;15;17),der(3)t(1;3;18),der(3)t(3;10)x2,+4,der(4)t(4;6),der(4)t(4;10)x2,der(4)t(4;12),+5,der(5)t(5;10;11;19),der(5)t(1;5;19),+10,+11,der(11)t(3;11),der(12)t(8;12),der(12)t(12;20),+14,der(14)t(14;16),der(14)t(14;21),+15,der(15)t(1;10;15;18),der(15)t(5;15;20), +17,der(17)t(3;15;17),der(17)t(13;17),+20,+21,der(X)t(2;X)x2. (+) denotes the gain or presence of extra copies of a specific chromosome, whereas (−) denotes the loss or deletion of a specific chromosome.

Most of these rearrangements were recurrent, appearing in the majority of metaphases analyzed, and were unbalanced, suggesting potential regional copy number alterations. In addition to these changes, chromosomes 20 and 21 were present in multiple copies in both cell lines. Consistent with previous reports (Pan et al., 1999), the Y chromosome was absent in PC-3 cells and similarly undetectable in PC-3 LCN2-KO cells. Importantly, chromosome 9, which contains the targeted LCN2 gene for knockdown, showed no rearrangement.

Both cell lines exhibit highly complex karyotypic rearrangements, with each rearranged chromosome comprising segments derived from multiple distinct chromosomes. In PC-3 cells (Figure 3), chromosome 1 (HSA1) is present in three copies, two of which are intact, while the third is rearranged and contains smaller segments originating from HSA8, HSA10, and HSA12. Additional HSA1 fragments are also detected on chromosomes HSA3 and HSA10. In contrast, in PC-3 LCN2-KO cells (Figure 4), HSA1 likewise occurs in three copies; however, only one copy remains intact, whereas the other two are extensively rearranged. Moreover, further HSA1-derived segments are observed on chromosomes HSA3, HSA5, and HSA15.

The pattern of rearrangements involving chromosome HSA3 is strikingly different between the two cell lines, with none of the chromosomes remaining intact. In PC-3 cells, all three copies of HSA3 contain translocated segments from HSA10. Additionally, in two identical large, complex rearranged chromosomes, segments from HSA1 and HSA10 are interspersed along both the short and long arms around the centromere. FISH experiments with single chromosome paints were carried out in metaphases of both lines to validate complex translocation as well as the SKY hybridization pattern (Figure 5).

Validation of SKY hybridization patterns by FISH using individual chromosome paints in both cell lines. Single-chromosome FISH results for (A) PC-3 and (B) PC-3 LCN2-KO are shown on the left, with the corresponding SKY images depicting the rearranged chromosomes on the right. (C) FISH with chromosome paints for chromosomes 1 and 10 in high resolution revealing interspersed segments within the rearranged complex chromosome 3, as shown in the SKY images. The adjacent schematic depicts the arrangement of each chromosomal segment (chromosome 1, colored in red, chromosome in DAPI stain in blue, and chromosome 10 in green).

In the knockout PC-3 LCN2-KO cells, HSA3 is present in more than three copies, most of which contain segments from HSA10. The large, complex translocated chromosome that comprises material from HSA3, HSA1, and HSA10 and is present in two copies in PC-3 cells is reduced to a single copy in the knockout cells. Additionally, four smaller rearranged copies of chromosome 3 were observed: one carrying segments from HSA1 and HSA18; and two smaller copies including segments from HSA10.

The rearrangement patterns of most HSA5 copies were largely similar between the two cell lines. However, one rearranged copy in PC-3 LCN2-KO included a segment derived from HSA1, whereas the corresponding region in PC-3 contained sequences from HSA15. Notably, a single rearranged HSA10 in PC-3 harbored two segments from HSA1 and one from HSA17, whereas in PC-3 LCN2-KO, both copies of HSA10 were involved in translocations across nine different rearranged chromosomes. These observations suggest that LCN2 knockout is associated with additional chromosomal rearrangements, particularly involving HSA10 and HSA5, indicating potential changes in genome stability in the knockout line.

Additional differences are observed in chromosomes HSA14, HSA15, and HSA17, which display specific rearrangements in PC-3 LCN2-KO. For HSA12, PC-3 cells exhibit segments from HSA3, HSA12, HSA15, and HSA17, whereas the knockout cells show only a single translocation between HSA8 and HSA12. A more detailed comparison of the rearrangement patterns between these two cell lines is highlighted in Table 1.

Frequency of translocationsPC-3PC-3 LCN2-KOCommonTranslocations with lower frequency in 20 metaphases (7-11x)t (17; 10; 1;10; 1;10)
t (3; 17; 15; 1;10)
t (3; 17; 15; 17)
t (8; 12)t (7; 18)—Translocations with higher frequency in 20 metaphases (12-16x)t (7; 11)t (3; 1;18)
t (15; 1;10; 18; 10; 1;15)
t (8; 12; 1;10)
t (5; 10; 11)
t (14; 16)—Major translocations (17-20x)t (5; 19; 15)
t (8; 12; 1;10)
t (5; 19; 10; 11)
t (14; 16)t (5; 19; 1)
t (11; 3)
t (3; 17; 15; 1;10)
t (3; 17; 15; 17)
t (8; 12)t (8; 2)
t (17; 15; 2)
t (3; 10; 1;10; 1;10; 1;3)
t (4; 6)
t (4; 10)
t (4; 12)
t (12; 20)
t (15; 5;20)
t (17; 13)
t (X; 2)

Frequency of translocations present in PC-3 and PC-3 LCN2-KO.

SKY analysis of PC-3 and PC-3 LCN2-KO cell lines revealed 24 structurally rearranged chromosomes in the majority of 20 metaphases, with 17 aberrations (71%) shared between the two lines, indicating a largely conserved karyotypic background. Notably, distinct translocations were unique to each line: t (3; 18), t (5; 19; 1), t (11; 3), and t (15; 1;10; 18; 10; 1;15) were exclusive to PC-3 LCN2-KO, whereas t (5; 19; 15), t (17; 10; 1;10; 10), and t (7; 11) were detected only in PC-3.

Most copies of HSA5 exhibited similar rearrangement patterns in both lines; however, one rearranged HSA5 in PC-3 LCN2-KO included a segment from HSA1, contrasting with HSA15 in the parental line. Strikingly, while a single HSA10 in PC-3 carried two segments from HSA1 and one from HSA17, both HSA10 copies in PC-3 LCN2-KO were redistributed across nine different rearranged chromosomes. These differences suggest that LCN2 knockout contributes to additional, non-random chromosomal rearrangements, particularly involving HSA10 and HSA5, reflecting altered genome stability.

Although recurrent rearrangements are highlighted in the table, additional sporadic aberrations were also observed among the 20 metaphases analyzed. In total, PC-3 LCN2-KO cells displayed a higher total number of chromosomal aberrations (69) compared to the parental PC-3 line (59) (Supplementary Table S5). Furthermore, a comparison of the frequencies of specific chromosomes involved in rearrangements among the 20 analyzed metaphases revealed that 11 different chromosomes were more frequently rearranged in PC-3 LCN2-KO cells, compared to only three chromosomes in the parental PC-3 line (Supplementary Table S6). This finding suggests that the knockout cells may have an increased propensity for chromosomal instability relative to the parental PC-3 cells.

Analysis of detectable translocated segments relative to the normal diploid complement revealed a significant overall chromosomal gain in both lines (Table 2). However, SKY analysis is unable to detect losses. Additionally, DAPI-stained interphase nuclei consistently displayed micronucleus formation, either attached to or adjacent to the main nucleus. The frequency of micronuclei was significantly higher in PC-3 LCN2-KO than in PC-3 (Supplementary Figure S2), indicating a continuous ongoing elimination of chromosomal fragments before metaphase and emphasizing enhanced genomic instability in the knockout cells.

PC-3ChromosomeCopy number (modal)
(See Figure 3)Loss or gain observed in PC-3a (# cells/total)Interpretation13–4eGain:
No gain, no loss:
Loss:9/10
1/10
0/10Gain23d,f,gGain:
No gain, no loss:
Loss:10/10
0/10
0/10Gain32f–3f,gGain:
No gain, no loss:
Loss:8/10
1/10
1/10Gain42d,gGain:
No gain, no loss:
Loss:0/10
9/10
1/10Unchanged52,5d,f,gGain:
No gain, no loss:
Loss:10/10
0/10
0/10Gain62,5dGain:
No gain, no loss:
Loss:9/10
1/10
0/10Gain73fGain:
No gain, no loss:
Loss:10/10
0/10
0/10Gain84dGain:
No gain, no loss:
Loss:10/10
0/10
0/10Gain92Gain:
No gain, no loss:
Loss:0/10
10/10
0/10Unchanged10b2,5e,f,gGain:
No gain, no loss:
Loss:10/10
0/10
0/10Gain113,5