Spatial transcriptomics identifies candidate stromal drivers of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the “reawakening” of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture. Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.


Introduction
Benign prostatic hyperplasia (BPH) is the nodular proliferation of epithelial and stromal elements within the transition zone of the prostate (1).BPH leads to prostate enlargement and lower urinary tract symptomsincluding urinary storage and voiding problems -that cause substantial morbidity in men over 50 years old (1).Current BPH treatments -including α-adrenoceptor antagonists and 5α-reductase inhibitors -are only partially effective and may not target the underlying pathologic processes, which are still incompletely known.
In the 1970s, Stanford pathologist John McNeal recorded detailed observations of BPH histology that informed a new hypothesis of BPH etiology (2,3).He noted that the tangent ducts bordering BPH nodules exhibited epithelial hyperplasia and neoductal growth exclusively on the side facing into the nodule (Figure 1A).This eccentric effect suggested exposure to stromal inductive processes known to occur in prostate embryogenesis and later shown to be critical to epithelial and duct growth by tissue recombination studies (4).Thus, McNeal proposed that BPH nodules arise by the "reawakening" of embryonic inductive activity by adult prostate stroma.Whether the diffusible factors of BPH stroma mirror those of prostate embryogenesis has remained largely unknown.
Studies of gene-knockout mice and of rat prostatic rudiments in culture have identified secreted factors that modulate prostate development and ductal branching morphogenesis (5,6), including fibroblast growth factors (FGFs), insulin-like growth factors (IGFs), transforming growth factors (TGFs), bone morphogenic proteins (BMPs), WNT proteins (WNTs), and Sonic Hedgehog (SHH).By extension, many of those same factors have been implicated in BPH (7).More recently, bulk tissue and single-cell transcriptome studies have enumerated the genes expressed in human BPH tissues and cells (8,9) but have lacked histologic spatial resolution.
Here, by spatial transcriptomics of human BPH nodules, we identify secreted factors expressed in the presumptive inductive stroma adjacent to hyperplastic branching ducts.Our findings provide insight Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy.Decades ago, John McNeal proposed that BPH originates with the "reawakening" of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis.Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes.The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium.In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma.Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture.Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.

Spatial transcriptomics identifies factors secreted by presumptive inductive BPH stroma.
McNeal observed that tangent ducts bordering BPH nodules exhibit epithelial hyperplasia (with taller columnar epithelial cells) and neoductal growth exclusively on the side facing into the nodule (Figure 1A) (3), suggestive of inductive signals originating in the adjacent stroma.For shorthand, we have dubbed these branching ductal structures "BPH hubs" because of their superficial resemblance to a wagon wheel or USB hub (Figure 1B).
To investigate McNeal's hypothesis, we first sought to profile and compare gene expression in the "inner" (inside the nodule) versus "outer" adjacent stroma of BPH hubs.By examining H&E-stained sections of whole-mount human prostates (obtained from patients with prostate cancer and mild to severe BPH symptoms), we identified 6 BPH hubs with the classic features described by McNeal (Hubs 1-6 in Figure 1, C-H, and Supplemental Table 1; supplemental material available online with this article; https:// doi.org/10.1172/jci.insight.176479DS1).Combining laser capture microdissection (LCM) (Figure 2, A-E) and RNASeq, we generated paired spatial transcriptomes of the 6 BPH hubs.By supervised analysis of the gene expression data, we then identified genes whose expression was significantly enriched in the inner or outer stroma of BPH hubs (Figure 2F and Supplemental Table 2).Here, we focus on transcripts enriched in the inner stroma, which would include candidate inductive factors.
By RNA in situ hybridization (RISH), we verified enriched expression of IGF1 and CXCL13 in the inner stroma adjacent to BPH hubs, including among hubs that were microdissected and transcriptionally profiled (exemplified by Hub-1 in Figure 3, A-D, and quantified in Supplemental Table 4), as well as in additional independent hubs (exemplified by Hub-7 in Figure 3, E-K).Importantly, enriched expression did not merely reflect an enrichment of prostate fibroblasts, since interstitial prostate fibroblasts -identified by expression of complement factor 7 (C7) (9) -were observed in both the inner and outer stroma (Figure 3, D, H, and K).
IGF1 and CXCL13 expression were not exclusive to the BPH hub-adjacent stroma.By examining the full prostate cross sections, we observed stromal IGF1 and CXCL13 expression elsewhere within BPH nodules, but always in association with adjacent epithelial hyperplasia and ductal branching (as observed for the BPH hubs) (examples in Supplemental Figure 1).
IGF1 and CXCL13 are coexpressed in BPH fibroblasts and may function as paracrine factors.IGF1 and CXCL13 are both expressed in the inner stroma of BPH hubs.We next asked whether they were both expressed by the same cell.By single-cell RNA-Seq of paired BPH-normal human prostate tissue, we identified a discrete population of BPH fibroblasts (Figure 4, A and B, and Supplemental Figure 2).The BPH fibroblasts expressed both IGF1 and CXCL13, as well as steroid 5 alpha-reductase 2 (SRD5A2) and androgen receptor (AR) (P ≤ 2.7 × 10 -5 , Hypergeometric test) (Figure 4, C and D).We verified coexpression of IGF1 with CXCL13, SRD5A2, and AR by RISH (Figure 4, E-J, and quantified in Supplemental Table 4).
Since IGF1 is expressed in hub-adjacent BPH stroma, we next asked whether its receptor -insulin-like growth factor 1 receptor (IGF1R) -was expressed in the hub ductal epithelium.Indeed, by 2-color RISH, we observed epithelial IGF1R expression adjacent to IGF1 + stroma (Figure 5, A, B,  4).Perhaps more surprisingly (given the studied role of CXCL13 in lymphocyte chemoattraction), we also observed epithelial expression of the CXCL13 receptor -CXC chemokine receptor 5 (CXCR5) -adjacent to CXCL13 + stroma (Figure 5, C and F).Although expressed at lower levels than IGF1R, CXCR5 expression was clearly visible in comparison with negative control probes (Figure 5D).
IGF1 is a growth factor known to promote cell proliferation (13).Consistent with that function, we observed by immunohistochemistry increased Ki-67 + (proliferating) epithelial cells in BPH hub epithelium adjacent to inner IGF1 + CXCL13 + stroma, in comparison with the outer IGF1 -CXCL13 -stroma (Figure 5, G and H, and Supplemental Figure 3) (P = 0.04, paired 2-sided Student's t test).CXCL13 may have more than one function, but it has been best characterized as a lymphocyte chemoattractant (11).Perhaps surprisingly then, we observed B and T cell infiltrates within both the inner CXCL13 + stroma and the outer CXCL13 -stroma (example shown in Figure 5I).
IGF1 but not CXCL13 is expressed in human embryonic prostate stroma.McNeal's hypothesis -that BPH represents a reawakening of embryonic patterns of stromal induction -would imply that IGF1 and CXCL13 are also expressed in human embryonic prostatic stroma.To investigate this possibility,  4).
we carried out RISH on human fetal prostate tissues (obtained from elective terminations) of 12-15 weeks of gestation, within the time frame of embryonic ductal branching (14).We observed marked IGF1 expression within the embryonic prostate stroma encircling the urethra (Figure 6, A and B), and adjacent to new ductal budding (Figure 6, D and E) and ductal branching (Figure 6, G and H), as well  4).JCI Insight 2024;9(1):e176479 https://doi.org/10.1172/jci.insight.176479as IGF1R expression in the prostatic epithelium (Figure 6, B, E, and H).In contrast, CXCL13 expression was not apparent in the embryonic prostate stroma, although low-level expression of CXCL13 and/or CXCR5 could be observed in a small minority of epithelial cells (Figure 6, C, F, and I).
Given the observed CXCR5 expression in BPH epithelium, we also sought to evaluate CXCL13 function on BPH epithelium.Since CXCL13 is not a known component of BPH-1 cell media or prostate organoid media, we evaluated the effect of CXCL13 addition on BPH-1 spheroid and BPH organoid culture.Physiologic concentrations of recombinant human CXCL13 (100 ng/mL) had no discernable impact on spheroid or organoid formation (Figure 7, D, E, I, and J).However, we note that expression of the CXCL13 receptor CXCR5, by quantitative reverse transcription PCR (qRT-PCR), was substantially lower compared with IGF1R in BPH-1 spheroids and BPH organoids (Supplemental Figure 5).

Discussion
Decades ago, McNeal observed that the tangent ducts bordering BPH nodules display epithelial hyperplasia and neoductal growth exclusively on the side facing into the nodule, suggesting a response to inductive From mouse knockout studies, IGF1 was previously linked to prostate development and ductal branching morphogenesis (10), and indeed IGF signaling has long been implicated in BPH (20).However, much of the evidence has been circumstantial, and uncertainty has remained even over which prostate cell type(s) produce (or respond) to IGFs.In one study, primary human BPH fibroblasts were found to express IGF2 but not IGF1 (21).In another study, some but not other primary human prostate fibroblast cultures (albeit none derived from BPH tissue) showed androgen-stimulated expression of IGF1 (22).Other studies have implicated IGF1 expression from prostate epithelial cells and even macrophages (23).Thus, importantly, we believe our study is the first to identify high IGF1 expression in human BPH fibroblasts at the site of IGF1R + epithelial hyperplasia and neoductal growth, providing "smoking gun" evidence of the key role of stromal IGF1 in BPH nodular morphogenesis.
Our finding of high stromal IGF1 expression adjacent to BPH hubs is notable, but equally striking is what we did not find.Other growth factors -including FGFs, TGFs, BMPs, WNTs, and SHH -also have reported roles in fetal/neonatal prostate development (5,6) and by extension have been implicated in BPH (1,7).Yet among those growth factors, our studies identified only IGF1 (and IGF2) as top enriched genes.Our findings singularly highlight IGF1 as a critical growth factor in BPH nodular proliferation and therefore as a persuasive target for therapy.Our results may also explain why hyperinsulinemia is a risk factor for BPH (24); high insulin levels can crossactivate the IGF1R (17).Nonetheless, other growth factors -including bone morphogenetic protein 5 (BMP5) that we previously found highly overexpressed in bulk BPH tissue (8) -likely contribute to BPH, albeit exhibiting less polarized expression in the inner stroma, or perhaps with activity at earlier stages of nodular growth.
We recently reported markedly elevated expression of CXCL13 in bulk BPH tissue, where it was also part of a 65-gene BPH stromal signature that correlated with BPH symptom severity ( 8), but it had until then never been implicated in BPH.CXCL13 has been characterized as a lymphocyte chemoattractant (11).

R E S E A R C H A R T I C L E
JCI Insight 2024;9(1):e176479 https://doi.org/10.1172/jci.insight.176479Nonetheless, in our preliminary studies, we observed lymphocyte infiltrates within both the inner and outer stroma.Notably, we observed expression of CXCR5 (the only known CXCL13 receptor) in BPH epithelium.This finding suggests the possibility that CXCL13 functions as a paracrine factor in the growth or some other characteristic of adjacent prostatic epithelium.While our studies in 3D culture did not reveal that function, future studies (including in vivo) should inform the function(s) of CXCL13 in BPH pathogenesis.
The top gene transcripts overexpressed in inner (presumptive inductive) stroma were enriched for secreted factors, as predicted by McNeal's hypothesis.Notably, their known functions suggest roles not only in epithelial induction (IGF1, IGF2), but also in the proliferation, differentiation, or function of other cell types, including immune cells (CXCL13) (11), endothelial cells (PROK1) (25), and peripheral neurons (EDN3) (26).This finding underscores that BPH nodular proliferation is more than merely epithelial and fibroblast proliferation, but rather encompasses the morphogenesis of diverse new tissue elements.The function of other top-ranked inner stroma gene transcripts (e.g., PTN, PLAC9) remains to be investigated.Future studies will also evaluate the protein expression of the above factors (which is notoriously challenging for secreted proteins).Also of future interest are the gene transcripts enriched within the outer stroma of hubs, which might include factors that constrain epithelial proliferation.Single-cell spatial approaches will also refine the list and cell type source of inductive factors, though we note that current approaches would have missed CXCL13, which is not included in existing catalog gene sets.
Our studies were motivated by McNeal's observations and hypothesis that BPH is the reawakening of embryonic inductive activity by adult prostate stroma.Though mesenchymal induction of prostatic epithelial development was elucidated in the mouse and rat, recent studies demonstrated that human urogenital sinus mesenchyme is also an inducer of prostatic epithelial development (27).It is therefore notable that our findings only partly support McNeal's hypothesis.IGF1 is expressed in BPH inner (inductive) stroma and in the stroma of human fetal prostates undergoing ductal branching morphogenesis.However, CXCL13 expression is absent from the stroma of those fetal prostates.Thus, in the most straightforward interpretation, BPH nodular proliferation is not simply the reactivation of embryonic inductive patterns.Future studies that employ single-cell profiling of active chromatin (e.g., by ATAC-seq) (28) may reveal the processes regulating IGF1 and CXCL13 expression in BPH fibroblasts, and whether these reflect normal embryonic patterns or focal responses, for example to hormonal changes, inflammation, or aging.
Our studies also have several therapeutic implications.Most notably, 2 small molecule IGF1R inhibitors, each with different off-target profiles (18,19), abolished BPH-1 spheroid and patient-derived BPH organoid growth in cell culture.While follow-up studies (including in vivo studies) are needed, our findings suggest the potential efficacy of IGF1R inhibitors as a disease-targeted approach in preventing or managing BPH and its associated lower urinary tract symptoms.In cancer clinical trials, IGF1R inhibitors have shown manageable toxicity (albeit with limited clinical benefit against many cancers) (29); thus, they may find utility in BPH, particularly if delivery or activity can be restricted to the prostate.Indeed, inhibition of mTOR (which functions downstream of IGF1R) was reported to reduce prostate size (30).And saw palmetto extract, an alternative BPH therapy, may modulate the IGF1 signaling pathway (31).
Also intriguing is that the BPH fibroblasts expressing IGF1 and CXCL13 also express SRD5A2 (5α-reductase) and AR.Currently, 5α-reductase inhibitors (e.g., finasteride and dutasteride) are a first-line therapy for BPH, where they inhibit the conversion of testosterone to the more potent dihydrotestosterone, leading to the involution of androgen-dependent prostatic epithelium and prostate shrinkage (26).Of note, during prostate development, androgens act primarily on embryonic mesenchyme to prompt the secretion of paracrine-acting epithelial growth factors (32).Thus, it will be of interest to determine whether 5α-reductase inhibitors also affect the AR + BPH fibroblast expression of IGF1, CXCL13, and/or other secreted/inductive factors.If so, then these therapies might prove "more targeted" to BPH disease mechanisms than currently recognized.

Methods
Prostate tissue specimens.Human BPH tissue specimens were obtained from radical prostatectomy cases (done for prostate cancer) at the Stanford Hospital, with IRB approval and patient informed consent.LCM studies were done using archived FFPE blocks, while single-cell RNA-Seq and patient-derived organoid studies used fresh BPH tissue.Patient age, prostate size, and International Prostate Symptom Score are listed in Supplemental Table 1.Human fetal prostate specimens were obtained from elective terminations done at UCSF, with IRB approval and patient informed consent.

Figure 1 .
Figure 1."BPH hubs" suggest an inductive stroma.(A) Tangent ducts at the periphery of BPH nodules display epithelial hyperplasia (with taller columnar epithelial cells) (pink arrow) and neoductal growth (green arrow) exclusively on the side facing into the nodule, implying an inductive inner stroma (yellow star) and noninductive outer stroma (blue star).Histologic image from J McNeal in The Urologic Clinics of North America (3) with with permission of the publisher.(B) For shorthand, we term these ductal structures "BPH hubs" for their similarity to a wagon wheel or USB hub (shown).(C-H) Six hubs (Hubs 1-6) that display the classic features noted by McNeal were selected for laser capture microdissection.Inner stroma (yellow star) and outer stroma (blue star) are indicated.Scale bar is 500 μm.

Figure 2 .
Figure 2. Spatial transcriptomics of BPH hubs nominates stromal inductive factors.(A-E) Laser capture microdissection (LCM) exemplified for Hub-5.(A) H&E stain.Inner stroma (yellow star) and outer stroma (blue star) are indicated; scale bar is 500 μm.Note, the H&E section shown in A is the same as that presented in Figure 1G.(B) Corresponding LCM scope image.(C) Annotated LCM scope image.The red (inner stroma) and blue (outer stroma) markings register spots for infrared welding laser and boundaries for ultraviolet cutting laser.(D) Captured outer stroma.(E) Captured inner stroma.(F) Volcano plot of RNA-Seq-identified gene transcripts significantly overexpressed in the inner versus outer stroma of the 6 microdissected hubs.FDR is plotted against log 2 expression fold-change.Select genes significantly overexpressed in the inner (inductive) stroma are indicated.

Figure 4 .
Figure 4. IGF1 and CXCL13 are coexpressed in BPH fibroblasts.(A-D) Single-cell RNA-Seq of paired BPH-normal human prostate tissue.(A) t-Distributed stochastic neighbor embedding (t-SNE) plot of combined BPH/normal prostate cells.Each dot represents an individual cell.Cell clusters (colored) are annotated by the expression of known cell type markers (e.g., LUM and DCN in fibroblasts).(B) Close-up of fibroblast cells contributed by BPH (purple) or normal prostate (green).(C) Fibroblast gene expression levels shown for IGF1, CXCL13, SRD5A2, and AR.Color bar depicts log 2 transcript counts per cell.(D) Cell coexpression (purple) of IGF1 with CXCL13, SRD5A2, or AR.(E-J) Two-color RNA in situ hybridization (RISH) of BPH tissue, exemplified for Hub-8, verifies cell coexpression of (E and H) IGF1 (blue) with CXCL13 (red); (F and I) IGF1 (blue) with SRD5A2 (red); and (G and J) IGF1 (blue) with AR (red).Arrows identify representative cells with dual staining; scale bar is 500 μm, and all figure insets are an additional 8.7× magnification.Note, 2-color RISH for IGF1/CXCL13, IGF1/SRD5A2, and IGF1/AR was conducted on 1 additional BPH hub (Supplemental Table4).

Figure 6 .
Figure 6.IGF1 but not CXCL13 is expressed in stroma of human fetal prostates undergoing branching morphogenesis.Shown are cross sections of human fetal prostates at gestational ages (A-C) 12 weeks showing the urethra, (D-F) 14.6 weeks showing ductal budding from the urethra, and (G-I) 15 weeks showing ductal branching.(A, D, and G) H&E stains; scale bar is 500 μm.Note, the large black circle in G is a bubble under the coverslip.(B, E, and H) Two-color RNA in situ hybridization (RISH) of IGF1 (blue) and IGF1R (red).(C, F, and I) Two-color RISH of CXCL13 (blue) and CXCR5 (red).Arrows mark occasional epithelial cells with low-intensity CXCL13 and/or CXCR5 expression; scale bar is 500 µm, and all figure insets are an additional 3.2×-4.5×magnification.