The 22Rv1 Prostate Cancer Cell Line Carries Mixed Genetic Ancestry: Implications for Prostate Cancer Health Disparities Research Using Pre-Clinical Models

Abstract

BACKGROUND

Understanding how biological factors contribute to prostate cancer (PCa) health disparities requires mechanistic functional analysis of specific genes or pathways in pre-clinical cellular and animal models of this malignancy. The 22Rv1 human prostatic carcinoma cell line was originally derived from the parental CWR22R cell line. Although 22Rv1 has been well characterized and used in numerous mechanistic studies, no racial identifier has ever been disclosed for this cell line. In accordance with the need for racial diversity in cancer biospecimens and recent guidelines by the NIH on authentication of key biological resources, we sought to determine the ancestry of 22RV1 and authenticate previously reported racial identifications for four other PCa cell lines.

METHODS

We used 29 established Ancestry Informative Marker (AIM) single nucleotide polymorphisms (SNPs) to conduct DNA ancestry analysis and assign ancestral proportions to a panel of five PCa cell lines that included 22Rv1, PC3, DU145, MDA-PCa-2b, and RC-77T/E.

RESULTS

We found that 22Rv1 carries mixed genetic ancestry. The main ancestry proportions for this cell line were 0.41 West African (AFR) and 0.42 European (EUR). In addition, we verified the previously reported racial identifications for PC3 (0.73 EUR), DU145 (0.63 EUR), MDA-PCa-2b (0.73 AFR), and RC-77T/E (0.74 AFR) cell lines.

CONCLUSIONS

Considering the mortality disparities associated with PCa, which disproportionately affect African American men, there remains a burden on the scientific community to diversify the availability of biospecimens, including cell lines, for mechanistic studies on potential biological mediators of these disparities. This study is beneficial by identifying another PCa cell line that carries substantial AFR ancestry. This finding may also open the door to new perspectives on previously published studies using this cell line.

INTRODUCTION

African American (AA) men and other men of African ancestry have the highest incidence and mortality rates of prostate cancer (PCa) in the world, resulting in the highest reported cancer health disparity.^1–10 We use the term “African American” recognizing that while race is a social construct, racial classification remains extremely useful for describing general patterns of national health and health disparities, as most U.S. health data are reported by self-identified race.¹ Recent studies have provided compelling evidence in support of the notion that PCa health disparities result from the interplay of multiple factors, including biological/genetic factors.^11–14 For instance, several recent studies have reported genomic differences between AA men with PCa and Caucasian or European American (EA) with PCa, suggesting a potential role for biological mediators in driving PCa mortality disparities.^14–27 Understanding how these mediators contribute to increased PCa mortality in AA men requires mechanistic functional studies in pre-clinical cellular and animal models of PCa.

Given the inherent difficulties in conducting mechanistic studies in primary PCa cells, including transfection issues and high degree of molecular variability as these primary cells propagate in vitro,^28–31 it is essential to have available a well-characterized, racially diverse, patient-derived cohort of immortalized cell lines representing different stages of PCa. Unfortunately, there is currently a lack of racial diversity in the human PCa cell lines that are commercially available for research. While there is over a dozen of commercially available human prostate cell lines of European ancestry that are representative of various prostate phenotypes such as normal and different PCa stages, there are only three cell lines identified as having African ancestry (Table I).^32–45 This limits the scope of in vitro studies addressing mechanistic events involving potential biological factors associated with PCa health disparities.

Table I.

Commonly Used Commercially Available Prostate Cell Lines

Cell Line	Site of Origin	Race/Ethnicity	Source	Reference
22Rv1	Primary xenograft	Not Available	ATCC	Sramkoski RM et al. 199932
DU145	Brain metastasis	Caucasian	ATCC	Stone KR et al. 197833
E006AA-hT	Primary tumor	African American	ATCC	Koochekpour S et al. 201434
LAPC-3	Prostate xenograft	Not Available	ATCC	Klein KA et al. 199735
LAPC-4	Lymph node metastasis	Not Available	ATCC	Klein KA et al. 199735
LNCaP	Lymph node metastasis	Caucasian	ATCC	Horoscewicz JS et al. 198336
MDA-PCa-2a	Bone metastasis	Black	ATCC	Navone NM et al. 199737
MDA-PCa-2b	Bone metastasis	Black	ATCC	Navone NM et al. 199737
PC3	Bone metastasis	Caucasian	ATCC	Kaighn ME et al. 197938
PrEC	Normal human primary prostate epithelial cells	Variable	Lonza	Not applicable
PrSC/WPMY-1	Normal prostate/stroma	Caucasian	ATCC, Lonza	Webber MM et al. 199939
PWR-1E	Normal prostate	Caucasian	ATCC	Webber MM et al. 199640
PZ-HPV-7	Normal prostate/epithelial	Caucasian	ATCC	Weijerman PC, et al. 199441
RWPE-1	Normal prostate	Caucasian, White	ATCC	Bello D et al. 199742
RWPE2	Normal prostate	Caucasian, White	ATCC	Bello D et al. 199742
RWPE2-W99	Normal prostate	Caucasian, White	ATCC	Bello D et al. 199742
VCaP	Vertebral metastasis	Caucasian	ATCC	Loberg RD et al. 200643
WISH-PC2	Neuroendocrine xenograft prostate small-cell carcinoma	Caucasian	ATCC	Pinthus JH et al. 200844
WPE1-NA22	Prostate	Caucasian, White	ATCC	Webber MM et al. 200145
WPE1-NB11	Normal prostate	Caucasian	ATCC	Webber MM et al. 200145
WPE1-NB14	Normal prostate/epithelial	Caucasian, White	ATCC	Bello D et al. 199742
WPE1-NB26	Normal prostate	Caucasian, White	ATCC	Webber MM et al. 200145

ATCC, American Type Culture Collection

Research into molecular and genetic mechanisms underlying PCa mortality disparities will be greatly advanced by the availability of pre-clinical cellular models of AA prostate tumors representing different stages of the disease.⁴⁶ There is a growing awareness of the need for biospecimens from AA populations that could be used for biomedical research, and an effort has been made by PCa researchers to develop AA prostate cell lines for cancer health disparities research. ^46–51 For example, RC-77N/E and RC-77T/E is a pair of African American prostate cell lines that were recently developed by one of the co-authors in this study (CY), and are available for research upon request.⁴⁶ While we recognize that the use of cell lines in cancer research has limitations, and that determinants of PCa health disparities may not be conclusively identified via in vitro studies, the use of racially diverse PCa cell lines in mechanistic studies may provide important clues into contributing biological mediators.

The 22Rv1 human prostatic carcinoma cell line was derived from a xenograft of CWR22R cells serially propagated in mice after castration-induced regression.^32,52 A number of biological characteristics were specified when 22Rv1 was derived, including epithelial lineage, DNA content, and cytogenetic data.³² The donor patient clinical information provided for the parental cell line CWR22R was the diagnosis of Stage D primary prostatic carcinoma with Gleason score of 9 advanced to osseous metastasis.⁵² While conducting studies using 22Rv1 cells we noticed that no racial identifier was ever released for this cell line or the parental CWR22R, and no subsequent DNA ancestry analysis has been conducted to identify its ethnicity.^32,52 Recently, the National Institutes of Health established guidelines concerning the authentication of key biological resources, including cell lines, in order to ensure the identity and validity of the resource.⁵³ This authentication applies to racial identity if the cell line is being used in biological studies relevant to cancer health disparities. In this context, we sought to determine the ancestry of 22Rv1 and authenticate, using genetic ancestry analysis, a few previously racially identified cell lines used in PCa research. These included the PCa cell lines MDA-PCa-2b and RC-77T/E, previously reported as Black or AA,^37,46 and the PC3 and DU145 cell lines, previously reported as Caucasian or EA.^33,38

To determine the ancestry of the 22Rv1 cell line we used a set of Ancestry Informative Markers (AIMs) validated as single nucleotide polymorphism (SNP) genotypes for population structure analyses.⁵⁴ PC3, DU145, MDA-PCa-2b, and RC-77T/E cell lines were included in the study to further validate their previously reported ethnicities and serve as controls for our SNP genotypes.^33,37,38,46 Our analysis confirmed predominant European (EUR) ancestry in PC3 and DU145 cells as well as predominant West African (AFR) genetic ancestry in the MDA-PCa-2b and RC-77T/E cells. However, the 22Rv1 cell line was found to carry substantial AFR genetic ancestry. The racial classification of 22Rv1 as a mixed ancestry cell line with substantial AFR genetic ancestry expands diversity within the existing pool of human PCa cell lines. This finding may offer a new perspective on previously published studies using this cell line.

MATERIALS AND METHODS

Cell Culture

Cell lines were purchased from American Type Culture Collection (ATCC) and grown in a humidified incubator with 5% CO₂ at 37°C. Cells were routinely tested for mycoplasma contamination using MycoAlert^™ PLUS Mycoplasma Detection Kit (Lonza, Cat. # LT07). PC3 (ATCC, Cat. # CRL-1435), DU145 (ATCC, Cat. # HTB-81), and 22Rv1 (ATCC, Cat. # CRL-2505) cell lines were cultured in RPMI 1640 medium (Corning, Cat. # 10-040-CV) supplemented with 10% fetal bovine serum (Corning, Cat. # 35010CV), penicillin-streptomycin (Corning, Cat. # 30001Cl), and gentamicin (Gibco, Cat. # 15710064) as recommended by the supplier. MDA-PCa-2b (ATCC, Cat. # CRL-2422) cell line was cultured in F-12K medium (ATCC, Cat. # 30-2004) supplemented with 20% fetal bovine serum (Corning, Cat. # 35010CV), cholera toxin (Sigma-Aldrich, Cat. # C8052), epidermal growth factor (Sigma-Aldrich, Cat. # E4127), o-phosphoethanolamine (Sigma-Aldrich, Cat. # P0503), hydrocortisone (Sigma-Aldrich, Cat. # H0888), selenious acid (ACROS Organics, Cat. # AC19887), bovine insulin (Sigma-Aldrich, Cat. # I6634), and penicillin-streptomycin as recommended by the supplier. 0.2% Normocin (Invivogen, Cat. # ANT-NR-1) was added to the medium for PC3, DU145, 22Rv1, and MDA-PCa-2b cell lines to prevent contamination by mycoplasma, bacteria, or fungi. The RC-77T/E cell line was cultured with keratinocyte serum-free medium (K-SFM) with L-glutamine supplemented with bovine pituitary extract and recombinant epidermal growth factor 1-53 (Life Technologies, Cat. # 17-005-042). Fungizone (0.4%, Gibco, Cat. # 15290018) was added to this medium to prevent contamination by yeast or multicellular fungi. To ensure proper cell attachment to culture plates, RC-77T/E cell line was cultured in 1% collagen-treated dishes (100mm × 20mm) (Life Technologies, Cat. # A1064401).

DNA Extraction

DNA was extracted using the QIAamp DNA mini kit (Qiagen, Valencia, CA, Cat. # 51104) following the manufacturer’s protocol. Briefly, cells were grown in a monolayer at confluency no greater than 5×10⁶. Medium was aspirated and cells were washed with Dulbecco’s phosphate-buffered saline (PBS) (Corning, Cat. # 21030CM) and then trypsinized with 0.25% trypsin (Corning, Cat. # 25-053-Cl) to detach them from the culture flask. Cells were then collected in appropriate medium, transferred to a 1.5 ml microcentrifuge tube, and centrifuged for 5 minutes at 300 × g. Supernatant was removed and cell pellet was resuspended in PBS to a final volume of 200 μl. After this, 20 μl Proteinase K and 200 μl of Qiagen buffer AL were added and the sample was incubated for 10 min at 56°C. Then, 200 μl of ethanol was added, and DNA was purified using the columns provided in the kit according to the manufacturer’s instructions. The samples yielded approximately 6 μg of DNA with an A₂₆₀/A₂₈₀ ratio of 1.7–1.9. The samples were diluted in distilled water and stored at -20°C. Samples were shipped to the UCLA Sequencing and Genotyping Core, Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles for subsequent SNP genotyping.

Selection of Ancestry Informative Marker (AIM) SNPs

We used 29 Ancestry Informative Marker (AIM) SNPs validated in a previous study involving determination of admixture proportions in human continental populations.⁵⁴ We selected the smallest subset of 24 AIMs and 5 additional SNPs from the previous study, as a cost-and time-effective strategy, to determine the ancestry of the above-mentioned PCa cell lines of interest. All the 29 selected SNPs exhibited large allele frequency differences between four continental populations- AFR, EUR, Amer-Indian (AMI), and East Asian (EAS) in the previous study.⁵⁴ We confirmed the allele frequency differences between these ancestral groups for each of the 29 AIM SNPs using the NCBI dbSNP database (https://www.ncbi.nlm.nih.gov/snp).

SNP Genotyping

SNP genotyping was done using the Fluidigm Biomark HD system (South San Francisco, CA) at the UCLA Genotyping core facility. SNPtype^™ assays and reagents for each of the SNPs were purchased from Fluidigm. DNA samples and assays were loaded onto GT 96*96 Dynamic array and processed per Fluidigm protocol. The genotyping calls were made using Fluidigm SNP genotyping software. Each DNA sample was analyzed in triplicate to ensure accuracy.

DNA Ancestry Analysis

Data for the current study was analyzed as part of a larger project involving 1078 DNA samples. Markers showing different alleles at very high frequency in distinct putative parental populations were used to efficiently distinguish different populations and for the inference of ancestry membership proportions. In this study, we used 29 selected unlinked AIM SNPs as 29 loci for the STRUCTURE software version 2.3.4, for providing an estimation of genetic ancestry.^55–56 Allele frequencies for the 29 AIM SNPs are provided in Table II. The software was installed and run locally after importing the SNP data for 1078 DNA samples in the form of a matrix where the data for samples were in rows and the loci were in columns (.txt file). For the determination of true K or the assumed number of populations, length of the Burnin period used was 5,000, the number of Markov Chain Monte Carlo (MCMC) replicates after the Burnins was 50,000 and K was set for 1–10 with 3 replicates for each K. True K was determined to be equal to 2~3 by L(K) (log likelihood of each K) method, using Structure Harvester with a zipped version of the results from STRUCTURE, as an input file.^57–58 After the determination of true K, STRUCTURE was run three times, without any prior population assignment for an admixture population using 100,000 as the length of the Burnin period and 500,000 as the number of MCMC replicates after the burnins, for K=2 and K=3. The missing value was represented as -9, the maximum value of ALPHA (ALPHAMAX) was 10.0 and the SD of proposal for updating ALPHA was 0.025, while rest of the parameters used were default. The three clusters obtained as an output from STRUCTURE were assigned EUR, AFR, and AMI ancestries by crosschecking the allele frequencies for AIMs of multiple DNA samples within each cluster against those found in NCBI dbSNP. The bar plot was generated by STRUCTURE for visualization of ancestral proportions in each sample. PC3 and DU145 cells along with MDA-PCa-2b and RC-77T/E cells served as controls for EUR and AFR ancestries, respectively, and aligned well with the results where K=3.

Table II.

Validated Ancestry Informative Marker Single Nucleotide Polymorphisms to Determine Ancestry Assignment of Prostate Cancer Cell Lines

	dbSNP ID	Chromosome Number	Nucleotide Change	Allele 1 frequency
				EUR	AFR
1	rs4891825	18	G/A	0.89	0.09
2	rs9530435	13	T/C	0.79	0.07
3	rs1040045	6	T/C	0.73	0.10
4	rs7554936	1	T/C	0.34	0.99
5	rs772262	12	G/A	0.06	0.87
6	rs3784230	14	T/C	0.58	0.00
7	rs260690	2	C/A	0.91	0.36
8	rs10007810	4	G/A	0.25	0.96
9	rs7657799	4	T/G	0.05	0.86
10	rs6548616	3	T/C	0.25	0.96
11	rs4908343	1	G/A	0.82	0.04
12	rs11652805	17	T/C	0.14	0.98
13	rs9522149	13	T/C	0.76	0.03
14	rs10108270	8	C/A	0.35	0.97
15	rs6451722	5	G/A	0.24	0.90
16	rs9319336	13	T/C	0.05	0.11
17	rs870347	5	T/G	0.95	0.95
18	rs7803075	7	G/A	0.29	1.00
19	rs2416791	12	G/A	0.08	0.94
20	rs6422347	5	G/A	0.10	0.89
21	rs7997709	13	T/C	0.06	0.23
22	rs 731257	7	G/A	0.09	0.02
23	rs 4918842	10	T/C	0.15	0.09
24	rs10496971	2	T/G	0.09	0.06
25	rs12629908	3	G/A	0.05	0.16
26	rs3907047	20	C/T	0.06	0.00
27	rs1513181	3	C/T	0.14	0.21
28	rs1471939	8	C/T	0.17	0.32
29	rs2125345	17	A/G	0.32	0.99

dbSNP, NCBI Single Nucleotide Polymorphism database; EUR, European; AFR, West African

RESULTS

Informative Ancestry Markers for Distinguishing Ancestral Proportions

An initial subset of 24 AIM SNPs was selected from a validated larger set of 128 SNPs previously identified to define critical genome candidate regions and used to characterize samples from diverse population groups (Table II).⁵⁴ This subset of 24 AIMs contained specific SNPs capable of distinguishing the following four continent populations: EUR, AFR, AMI, and EAS.⁵⁴ Five additional AIMs from the validated larger set of 128 AIMs were added to the smaller subset of 24 AIMs, bringing our total quantity of AIMs used for DNA ancestry analysis to 29 (Table II).

Determination of Ancestral Proportions of Prostate Cancer Cell Lines

Each PCa cell line analyzed in our study possessed heterogeneous ancestral proportions (Figure 1, Table III). However, for each cell line where ATCC provided a racial classification, we observed a predominant ancestral proportion that was consistent with what was previously reported. For instance, PC3 and DU145 cell line samples were included in our analysis as EUR controls, and MDA-PCa-2b and RC-77T/E as AFR controls based on their previously reported racial classification.^33,37,38,46 PC3 is classified by ATCC as Caucasian and was found to carry 0.73 proportion of EUR ancestry. This cell line was also found to carry 0.19 AFR and 0.08 AMI ancestral proportions. DU145 is also classified by ATCC as Caucasian and was found to carry 0.63 proportion of EUR ancestry. Interestingly, DU145 was also found to carry 0.28 AFR and 0.09 AMI ancestral proportions. MDA-PCa-2b is classified by ATCC as Black and was found to carry 0.73 proportion of AFR ancestry as well as 0.13 AMI and 0.14 EUR ancestral proportions. RC-77T/E is not available through ATCC, however it was reported by one of the authors of this study (C. Yates) as an AA cell line,⁴⁶ and our results confirmed this. RC-77T/E was found to carry 0.74 proportion of AFR ancestry as well as 0.09 AMI and 0.17 EUR ancestral proportions. To date, there has never been a racial classification assigned to 22Rv1. Our results indicated the following ancestral proportions for 22Rv1: 0.41 AFR, 0.17 AMI, and 0.42 EUR. Therefore, 22Rv1 should be considered as a mixed ancestry cell line.

Figure 1. DNA Ancestry Analysis Reveals Ancestral Proportions of Prostate Cancer Cell Lines.

DNA was isolated and extracted from five PCa cell lines and submitted to the UCLA genomics core for SNP genotyping. Cell lines included EUR controls PC3 and DU145, AFR controls MDA-PCa-2b and RC-77T/E, and the racially ambiguous 22Rv1. DNA samples were analyzed in triplicate to ensure accuracy. The bar plot shown in this figure was generated using STRUCTURE for visualization of the ancestral proportion in each sample. Subsequent analysis of population substructure was also conducted using STRUCTURE, a model-based clustering software able to detect the underlying genetic population among a set of individuals genotyped at multiple markers with the help of a bayesian approach (http://pritch.bsd.uchicago.edu/software/structure2).⁵⁷ Using STRUCTURE, we were able to compute the proportion of the genome of the cell lines originating from each inferred population. Blue: AFR ancestry; Green: AMI ancestry; Red: EUR ancestry.

Table III.

Ancestry Proportions in Panel of Prostate Cancer Cell Lines

Cell Line	Proportion of Ancestry
	AFR	AMI	EUR
PC3	0.19	0.08	0.73
DU145	0.28	0.09	0.63
MDA-PCa-2b	0.73	0.13	0.14
RC-77T/E	0.74	0.09	0.17
22Rv1	0.41	0.17	0.42

Samples were analyzed in triplicates to ensure accuracy.

AFR, African ancestry; AMI, Ameri-Indian ancestry; EUR, European ancestry.

DISCUSSION

In concordance with recent NIH guidelines requiring authentication of cell lines, this study sought to determine the racial identity of 22Rv1 PCa cells since racial classification/ethnicity for this cell line has never been reported to ATCC or previously published.^32,52,53 It has been our experience in personal communication with other groups that that 22Rv1 is widely considered as a EA PCa cell line, and we had no reason to explore this assumption until we began incorporating various EA and AA cell lines in our studies.^27,59,60 We initially conducted a thorough literature search and contacted ATCC to determine if there was any indication of racial identification for either 22Rv1 or its parental cell line CWR22R. In addition, we communicated with key authors of the initial reports on the characterization of both cell lines to determine if there was any racial identification linked to the tumor biospecimen from which these cell lines were derived.^32,52 Unfortunately, these efforts did not provide any indication as to the racial classification of these cell lines. This lack of information prompted us to conduct DNA ancestry analysis on 22Rv1 cells and provide this information to the PCa research community and the scientific community at large. We felt this was a very important issue given the need for a comprehensive panel of racially diverse patient-derived cell lines for mechanistic studies on potential biological mediators of PCa mortality disparities.

SNP genotyping and subsequent DNA ancestry analysis were chosen as appropriate methods to determine the ancestry for 22Rv1. Other investigators have shown that differences in allele frequencies can be used to identify continental population groups.^61,62 This is because several studies have demonstrated that large differences in allele frequencies between two or more continental populations can be attributed to many individual SNPs distributed throughout the genome.^63–67 In this study, 29 AIM SNPs were chosen from a validated larger set of 128 SNPs previously identified by others to define critical genome candidate regions and used to characterize samples from diverse population groups.⁵⁴ Differences in population structure were addressed by extending genetic associations to minority groups that include extensive admixture between continents.⁵⁴ This genetic association extension was essential to evaluate ethnic disparities in disease risk among multiethnic and admixed populations as a large number of whole genome association studies have evaluated populations of predominantly EUR ancestry.⁵⁴

Our analysis revealed that the 22Rv1 PCa cell line carries AFR genetic ancestry, with almost equal proportions for AFR and EUR ancestry (41% and 42%, respectively). While this study authenticates the previously reported racial identification of several PCa cell lines (PC3, DU145, MDA-PC-2b, and RC-77T/E), it establishes for the first time the ancestral composition of 22Rv1. Because 22Rv1 was derived from a xenograft of CWR22R which was serially propagated in mice,^32,52 it would be appropriate to also assign this mixed ancestral racial classification to CWR22R. The implications of 22Rv1 and CWR22R carrying AFR genetic ancestry are far reaching as genetic factors associated with AFR ancestry have been recently linked to prostate tumor aggressive properties.^15–26 Further studies are needed to determine if these cell lines carry any of these factors. In addition, our findings open the door for closely examining previous studies using 22Rv1 and CWR22R cells under the new perspective that these cell lines carry almost equal proportions of AFR and EUR genetic ancestry.

We suggest that 22Rv1 and CWR22R can be be added to the current roster of PCa cell lines that are classified as Black, AA, or carry substantial AFR genetic ancestry, including MDA-PCa-2b, E006AA-hT, RC-77N/E, and RC-77T/E.^33,36,45 Alternatively, they could be classified in a separate category as racially mixed ancestry cell lines. We cannot rule out the possibility that CWR22R was originally derived from a Hispanic male of Caribbean heritage as the proportions of AFR, AMI, and EUR genetic ancestry found in the 22RV1 cell line are common in men from this geographical region.^68–70 For example, recent studies have highlighted the high tri-hybrid admixture (Native American, European, West African) of the Puerto Rican population.^68–69 Knowledge that 22Rv1 carries substantial AFR ancestry would be beneficial to efforts to expand the selection of racially diverse cell lines for in vitro mechanistic studies focusing on potential determinants PCa mortality disparities that could be attributable to genetically- and racially-driven tumor biological differences. It is important to note that while this study contributes to the increasing diversity of PCa cell lines, thus far all available AA PCa cell lines are derived from patients with metastatic disease and tumors with functional androgen and glucocorticoid receptors. There remains a critical need for AA PCa cell lines derived from metastatic androgen-independent tumors (e.g. equivalent to the EA PC3 and DU145). This would be important for mechanistic studies on different disease stages using AA PCa cell lines.

CONCLUSIONS

The field of PCa health disparities research continues to advance, with recent attention to potential biological determinants or mediators of these disparities that could be mechanistically examined using a panel of racially diverse, patient-derived cell lines. The identification of 22Rv1 as a mixed ancestry PCa cell line carrying substantial AFR genetic ancestry provides an additional resource to advance molecular/cellular research in the context of PCa health disparities. While there still remains a deficit in the availability of AA PCa cell lines and clinical biospecimens, the trend towards acquiring additional specimens appears to be growing.^47–51 The knowledge to be gained from mechanistic studies incorporating biospecimens (cell lines plus tumor samples) from AA men will not only have the potential to reduce the mortality disparities associated with this malignancy but also benefit the field of PCa research in general.