Estrogen receptor α in cancer associated fibroblasts suppresses prostate cancer invasion via reducing CCL5, IL6 and macrophage infiltration in the tumor microenvironment.

BACKGROUND: Cancer associated fibroblasts (CAF) play important roles in tumor growth that involves inflammation and epithelial cell differentiation. Early studies suggested that estrogen receptor alpha (ERα) was expressed in stromal cells in normal prostates and prostate cancer (PCa), but the detailed functions of stromal ERα in the PCa remain to be further elucidated. METHODS: Migration and invasion assays demonstrated the presence of high levels of ERα in CAF cells (CAF.ERα(+)) suppressed PCa invasion via influencing the infiltration of tumor associated macrophages. ERα decreased CAF CCL5 secretion via suppressing the CCL5 promoter activity was examined by luciferase assay. ERα decreased CCL5 and IL-6 expression in conditioned media that was collected from CAF cell only or CAF cell co-cultured with macrophages as measured by ELISA assay. RESULTS: Both in vitro and in vivo studies demonstrated CAF.ERα(+) led to a reduced macrophage migration toward PCa via inhibiting CAF cells secreted chemokine CCL5. This CAF.ERα(+) suppressed macrophage infiltration affected the neighboring PCa cells invasion and the reduced invasiveness of PCa cells are at least partly due to reduced IL6 expression in the macrophages and CAF. CONCLUSION: Our data suggest that CAF ERα could be applied as a prognostic marker to predict cancer progression, and targeting CCL5 and IL6 may be applied as an alternative therapeutic approach to reduce M2 type macrophages and PCa invasion in PCa patients with low or little ERα expression in CAF cells.

Quantitative volumetric imaging of normal, neoplastic and hyperplastic mouse prostate using ultrasound.

BACKGROUND: Genetically engineered mouse models are essential to the investigation of the molecular mechanisms underlying human prostate pathology and the effects of therapy on the diseased prostate. Serial in vivo volumetric imaging expands the scope and accuracy of experimental investigations of models of normal prostate physiology, benign prostatic hyperplasia and prostate cancer, which are otherwise limited by the anatomy of the mouse prostate. Moreover, accurate imaging of hyperplastic and tumorigenic prostates is now recognized as essential to rigorous pre-clinical trials of new therapies. Bioluminescent imaging has been widely used to determine prostate tumor size, but is semi-quantitative at best. Magnetic resonance imaging can determine prostate volume very accurately, but is expensive and has low throughput. We therefore sought to develop and implement a high throughput, low cost, and accurate serial imaging protocol for the mouse prostate. METHODS: We developed a high frequency ultrasound imaging technique employing 3D reconstruction that allows rapid and precise assessment of mouse prostate volume. Wild-type mouse prostates were examined (n = 4) for reproducible baseline imaging, and treatment effects on volume were compared, and blinded data analyzed for intra- and inter-operator assessments of reproducibility by correlation and for Bland-Altman analysis. Examples of benign prostatic hyperplasia mouse model prostate (n = 2) and mouse prostate implantation of orthotopic human prostate cancer tumor and its growth (n = ) are also demonstrated. RESULTS: Serial measurement volume of the mouse prostate revealed that high frequency ultrasound was very precise. Following endocrine manipulation, regression and regrowth of the prostate could be monitored with very low intra- and interobserver variability. This technique was also valuable to monitor the development of prostate growth in a model of benign prostatic hyperplasia. Additionally, we demonstrate accurate ultrasound image-guided implantation of orthotopic tumor xenografts and monitoring of subsequent tumor growth from ~10 to ~750 mm(3) volume. DISCUSSION: High frequency ultrasound imaging allows precise determination of normal, neoplastic and hyperplastic mouse prostate. Low cost and small image size allows incorporation of this imaging modality inside clean animal facilities, and thereby imaging of immunocompromised models. 3D reconstruction for volume determination is easily mastered, and both small and large relative changes in volume are accurately visualized. Ultrasound imaging does not rely on penetration of exogenous imaging agents, and so may therefore better measure poorly vascularized or necrotic diseased tissue, relative to bioluminescent imaging (IVIS). CONCLUSIONS: Our method is precise and reproducible with very low inter- and intra-observer variability. Because it is non-invasive, mouse models of prostatic disease states can be imaged serially, reducing inter-animal variability, and enhancing the power to detect small volume changes following therapeutic intervention.

Estrogen receptor α in cancer-associated fibroblasts suppresses prostate cancer invasion via modulation of thrombospondin 2 and matrix metalloproteinase 3.

The prostate cancer (PCa) microenvironment contains active stromal cells known as cancer-associated fibroblasts (CAF) that may play important roles in influencing tumor progression. Here we studied the role of CAF estrogen receptor alpha (ERα) and found that it could protect against PCa invasion. Immunohistochemistry on prostatectomy specimens showed that PCa patients with ERα-positive stroma had a significantly lower risk for biochemical recurrence. In vitro invasion assays further confirmed that the stromal ERα was able to reduce PCa cell invasion. Dissection of the molecular mechanism revealed that the CAF ERα could function through a CAF-epithelial interaction via selectively upregulating thrombospondin 2 (Thbs2) and downregulating matrix metalloproteinase 3 (MMP3) at the protein and messenger RNA levels. Chromatin immunoprecipitation assays further showed that ERα could bind to an estrogen response element on the promoter of Thbs2. Importantly, knockdown of Thbs2 led to increased MMP3 expression and interruption of the ERα mediated invasion suppression, providing further evidence of an ERα-Thbs2-MMP3 axis in CAF. In vivo studies using athymic nude mice injected with CWR22Rv1 (22Rv1) PCa epithelial cells and CAF cells ± ERα also confirmed that mice coimplanted with PCa cells and CAF ERα+ cells had less tumor foci in the pelvic lymph nodes, less metastases, and tumors showed less angiogenesis, MMP3, and MMP9 (an MMP3 downstream target) positive staining. Together, these data suggest that CAF ERα could play protective roles in suppressing PCa metastasis. Our results may lead to developing new and alternative therapeutic approaches to battle PCa via controlling ERα signaling in CAF.

Strain-dependent glutathionylation of fibronectin fibers impacts mechano-chemical behavior and primes an integrin switch.

The extracellular matrix (ECM) is a protein polymer network that physically supports cells within a tissue. It acts as an important physical and biochemical stimulus directing cell behaviors. For fibronectin (Fn), a predominant component of the ECM, these physical and biochemical activities are inextricably linked as physical forces trigger conformational changes that impact its biochemical activity. Here, we analyze whether oxidative post-translational modifications, specifically glutathionylation, alter Fn's mechano-chemical characteristics through stretch-dependent protein modification. ECM post-translational modifications represent a potential for time- or stimulus-dependent changes in ECM structure-function relationships that could persist over time with potentially significant impacts on cell and tissue behaviors. In this study, we show evidence that glutathionylation of Fn ECM fibers is stretch-dependent and alters Fn fiber mechanical properties with implications on the selectivity of engaging integrin receptors. These data demonstrate the existence of multimodal post-translational modification mechanisms within the ECM with high relevance to the microenvironmental regulation of downstream cell behaviors.

Loss of epithelial oestrogen receptor α inhibits oestrogen-stimulated prostate proliferation and squamous metaplasia via in vivo tissue selective knockout models.

Squamous metaplasia (SQM) is a specific phenotype in response to oestrogen in the prostate and oestrogen receptor (ER) α is required to mediate this response. Previous studies utilizing tissue recombination with seminal vesicle (SV) mesenchyme and prostatic ductal tips from wild type and ERαKO mice suggested that both epithelial and stromal ERα are necessary for SQM. However, tissue recombination is conducted in the renal capsule of immune-deficient mice, in which the microenvironment is different from normal prostate microenvironment in the intact mice. Furthermore, whether the requirement of stromal ERα in the SV for developing SQM is the same as in the prostate is unknown. Therefore, there is a clear need to evaluate the respective roles of ERα in prostate epithelial versus stromal compartments in the intact mouse. Here we generated a mouse model that has selectively lost ERα in either stromal (FSP-ERαKO) or epithelial prostate cells (pes-ERαKO) to determine the requirements of ERα for oestrogen-stimulated prostate proliferation and SQM. Our results indicated that FSP-ERαKO prostates develop full and uniform SQM, which suggests that loss of the majority (~65%) of stromal ERα will not influence oestrogen-mediated SQM. In contrast, loss of epithelial ERα inhibits oestrogen-mediated prostate growth and SQM evidenced by decreasing cytokertin 10 positive squamous cell stratification and differentiation, by reduced ERα protein expression in SQM compared to wild type mice ERα, and by the presence of normal proliferative activities in the oestrogen-treated pes-ERαKO prostates. These in vivo results suggest that epithelial ERα is required for oestrogen-mediated proliferative response and could be an appropriate target for preventing aberrant oestrogen signalling in the prostate.

SEMA7a primes integrin α5ß1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming.

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5ß1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

BACKGROUND: Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF-1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear. METHODS: To further probe the stromal fibroblast AR roles in prostate development, we generated tissue-selective knockout mice with the AR gene deleted in stromal fibroblasts (FSP-ARKO). We also used primary culture stromal cells to confirm the in vivo data and investigate mechanisms related to prostate development. RESULTS: The results showed cellular alterations in the FSP-ARKO mouse prostate with decreased epithelial proliferation, increased apoptosis, and decreased collagen composition. Further mechanistic studies demonstrated that FSP-ARKO mice have defects in the expression of prostate stromal growth factors. To further confirm these in vivo findings, we prepared primary cultured mouse prostate stromal cells and found knocking down the stromal AR could result in growth retardation of prostate stromal cells and co-cultured prostate epithelial cells, as well as decrease of some stromal growth factors. CONCLUSIONS: Our FSP-ARKO mice not only provide the first in vivo evidence in Cre-loxP knockout system for the requirement of stromal fibroblast AR to maintain the normal development of the prostate, but may also suggest the selective knockdown of stromal AR might become a potential therapeutic approach to battle prostate hyperplasia and cancer.

Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad.

Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell growth and metastasis. We investigated the roles of flow-induced shear stress in modulating cell cycle distribution in four tumor cell lines and the underlying mechanisms. In all four cell lines, incubation under static conditions for 24 or 48 h led to G(0)/G(1) arrest; in contrast, shear stress (12 dynes/cm(2)) induced G(2)/M arrest. The molecular basis of the shear effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 osteosarcoma cells. Shear stress induced increased expressions of cyclin B1 and p21(CIP1) and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27(KIP1) as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G(2)/M arrest and corresponding changes in G(2)/M regulatory protein expression and activity were mediated by alpha(v)beta(3) and beta(1) integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expressions in MG63 cells; these responses were mediated by alpha(v)beta(3) and beta(1) integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G(2)/M arrest in tumor cells and inhibits cell differentiation and demonstrate the importance of mechanical microenvironment in modulating molecular signaling, gene expression, cell cycle, and functions in tumor cells.

Decellularized Extracellular Matrix (ECM) as a Model to Study Fibrotic ECM Mechanobiology.

Aberrant deposition of the extracellular matrix (ECM) causes fibrosis and leads to ECM stiffening. This fibrotic ECM provides biological and biophysical stimulations to alter cell activity and drive progression of fibrosis. As an emerging discipline, mechanobiology aims to access the impact of both these cues on cell behavior and relates the reciprocity of mechanical and biological interactions; it incorporates concepts from different fields, like biology and physics, to help study the mechanical and biological facets of fibrosis extensively. A useful experimental platform in mechanobiology is decellularized ECM (dECM), which mimics the native microenvironment more accurately than standard 2D culture techniques as its composition includes similar ECM protein components and stiffness. dECM, therefore, generates more reliable results that better recapitulate in vivo fibrosis.

Citrullination of fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion.

The extracellular matrix (ECM) microenvironment is increasingly implicated in the instruction of pathologically relevant cell behaviors, from aberrant transdifferentation to invasion and beyond. Indeed, pathologic ECMs possess a panoply of alterations that provide deleterious instructions to resident cells. Here we demonstrate the precise manner in which the ECM protein fibronectin (FN) undergoes the posttranslational modification citrullination in response to peptidyl-arginine deiminase (PAD), an enzyme associated with innate immune cell activity and implicated in systemic ECM-centric diseases, like cancer, fibrosis and rheumatoid arthritis. FN can be citrullinated in at least 24 locations, 5 of which reside in FN's primary cell-binding domain. Citrullination of FN alters integrin clustering and focal adhesion stability with a concomitant enhancement in force-triggered integrin signaling along the FAK-Src and ILK-Parvin pathways within fibroblasts. In vitro migration and in vivo wound healing studies demonstrate the ability of citrullinated FN to support a more migratory/invasive phenotype that enables more rapid wound closure. These findings highlight the potential of ECM, particularly FN, to "record" inflammatory insults via post-translational modification by inflammation-associated enzymes that are subsequently "read" by resident tissue fibroblasts, establishing a direct link between inflammation and tissue homeostasis and pathogenesis through the matrix.

Estrogen receptor ß promotes renal cell carcinoma progression via regulating LncRNA HOTAIR-miR-138/200c/204/217 associated CeRNA network.

Recent studies indicated that the estrogen receptor beta (ERß) could affect the progression of prostate and bladder tumors, however, its roles in the renal cell carcinoma (RCC), remain to be elucidated. Here, we provide clinical evidence that ERß expression is correlated in a negative manner with the overall survival/disease-free survival in RCC patients. Mechanism dissection revealed that targeting ERß with ERß-shRNA and stimulating the transactivation of ERß with 17ß-estradiol or environmental endocrine disrupting chemicals, all resulted in altering the lncRNA HOTAIR expression. The ERß-modulated HOTAIR is able to function via antagonizing several microRNAs, including miR-138, miR-200c, miR-204, or miR-217 to impact various oncogenes, including ADAM9, CCND2, EZH2, VEGFA, VIM, ZEB1, and ZEB2, to promote RCC proliferation and invasion. Together, the identification of the ERß-HOTAIR axis may provide us new biomarkers and/or therapeutic targets to better suppress RCC progression in the future.

Targeting newly identified ERß/TGF-ß1/SMAD3 signals with the FDA-approved anti-estrogen Faslodex or an ERß selective antagonist in renal cell carcinoma.

Renal cell carcinoma (RCC) has the third highest mortality rate among urological tumors, and 20-30% of RCC patients present with metastatic RCC at the time of diagnosis. Although recent studies have indicated that estrogen receptor ß (ERß) could play promoting roles in RCC progression, the detailed mechanisms remain to be clarified. In the present study, we found that expression of ERß, but not ERα, increases with tumor stage and grade, and also observed that modification of ERß signals using estrogens/anti-estrogens, shRNA knockdown of ERß and overexpression of ERß using ectopic cDNA affects RCC cell proliferation, migration and invasion. Mechanism analysis revealed that ERß can promote RCC cell invasion via an increase in transforming growth factor ß1 (TGF-ß1)/SMAD3 signals, and interrupting TGF-ß1/SMAD3 signals with a TGFßR1 inhibitor can reverse/block ERß-increased RCC cell migration. Importantly, preclinical analyses using in vivo mouse models of RCC revealed that targeting of this newly identified ERß/TGF-ß1/SMAD3 pathway with either the FDA-approved anti-estrogen ICI182,780 (Faslodex) or a selective ERß antagonist 4-[2-phenyl-5,7 bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol can significantly reduce RCC tumor growth and invasion, which may be suitable as the basis for novel therapies to more effectively suppress metastatic RCC.

Recruited mast cells in the tumor microenvironment enhance bladder cancer metastasis via modulation of ERß/CCL2/CCR2 EMT/MMP9 signals.

Early clinical studies suggested that infiltrating mast cells could be associated with a poor outcome in bladder cancer (BCa) patients. The mechanisms of how mast cells influence the BCa progression, however, are unclear. Using the human clinical BCa sample survey and in vitro co-culture systems, we found BCa cells could recruit more mast cells than the surrounding non-malignant urothelial cells. The consequences of this better recruitment of mast cells toward BCa cells could then enhance BCa cell invasion. Mechanism dissection revealed that the enhanced BCa cell invasion could function via up-regulation of the estrogen receptor beta (ERß) in both mast cells and BCa cells, which resulted in the increased CCL2/CCR2/EMT/MMP9 signals. Using the pre-clinical mouse BCa model, we further validated the mast cell-promoted BCa invasion. Interruption of the newly identified ERß/CCL2/CCR2/EMT/MMP9 pathway via either ERß-siRNA, ERß antagonist PHTPP, or CCR2 antagonist can effectively reverse the mast cell-enhanced BCa cells invasion. Together, our finding could lead to the development of an alternative new therapeutic approach to better treat BCa metastasis.

The influence of mechanical properties of subchondral plate, femoral head and neck on dynamic stress distribution of the articular cartilage.

A few finite element models have addressed the dynamic juxtaarticular stress transmission but none focused the investigation on the combined influence of the individual moduli of the underlying bones, including the subchondral plate, the femoral head and the femoral neck of the proximal femur. A finite element model including the acetabulum and the proximal femur was analyzed with dynamic loadings to study the effects of mechanical property changes in the underlying bones of the proximal femur on the stress distribution in the cartilage at the hip joint. We found the stress distribution was most sensitive to the subchondral plate stiffening, while the overall stiffening of the underlying bones had mild effect on the shear stress on the cartilage surface (or at the subchondral bone/cartilage interface) and on the strain energy density in the cartilage. Our results indicate that the subchondral plate plays a predominant mechanical role in the initial degeneration of the cartilage. The results may offer a mechanical explanation as to why the cartilage failure is common in patients with osteoarthritis but rare in patients with osteoporosis.

Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor ß-DAB2IP signals.

Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERß), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERß expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERß and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERß expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERß transcriptional activity. The newly identified relationship between infiltrating T cells/ERß/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

Infiltrating neutrophils promote renal cell carcinoma progression via VEGFa/HIF2α and estrogen receptor ß signals.

Neutrophils make up a significant portion of the infiltrated immune cells found in the tumor microenvironment. Here we found more infiltrated neutrophils in human renal cell carcinoma (RCC) lesions than adjacent benign areas. In vitro RCC studies showed that neutrophils (HL-60N cells) infiltrated toward RCC cells and subsequently enhanced RCC cell migration and invasion. Co-culture of RCC cells with HL-60N cells up-regulated ERß, VEGFa and HIF2α mRNA levels. ERß signals increased RCC cell migration via induction of the VEGFa/HIF2α pathway. Treatment of HIF inhibitor or rapamycin, or knockdown of ERß in RCC cells reversed HL-60N-promoted RCC migration. In vivo data using orthotopically xenografted RCC mouse model confirmed that infiltrated neutrophils promoted RCC migration via modulating the expressions of ERß, VEGFa and HIF2α signal pathways. Together, our studies revealed that neutrophils are favorably recruited to the RCC cells to promote the RCC migration and invasion. Targeting the infiltrating RCC tumor microenvironment with anti-estrogen or rapamycin may be a potential therapy to suppress RCC progression.

Fibroblast ERα promotes bladder cancer invasion via increasing the CCL1 and IL-6 signals in the tumor microenvironment.

Epidemiological studies indicate that women have a higher chance of developing muscle invasive bladder cancer (BCa) than men, suggesting that estrogen and estrogen receptors (ERs) may play critical roles in BCa progression. However, the ERs roles in the bladder tumor microenvironment and impacts on BCa progression remain largely unclear. Using IHC staining in human BCa samples, we found that higher ERα expression in the stromal compartment of BCa may be correlated with unfavorable clinical outcomes. Results from cell line studies revealed that co-culturing with fibroblasts could promote BCa T24, UMUC3 and 5637 cells invasion. Importantly, addition of ERα in fibroblasts further enhanced the BCa cell invasion and knock-down of ERα in fibroblasts could then partially reduce the fibroblasts-enhanced BCa invasion. Mechanism dissection suggested that ERα could function through modulating the CCL cytokines expression in fibroblasts to increase the BCa IL-6 expression. An interruption approach using IL-6 neutralizing antibody then reversed the fibroblast ERα-enhanced BCa cell invasion. Together, these data suggest that the higher expression of ERα in fibroblasts may be the result of modulating the CCL1 expression in fibroblasts and/or IL-6 production in BCa cells to enhance BCa cells invasion. Targeting these individual molecules in this newly identified ERα-stimulated CCL1 and IL-6 signal pathways may become an alternative therapy to better suppress the BCa cell invasion.

Infiltrating neutrophils promote renal cell carcinoma (RCC) proliferation via modulating androgen receptor (AR) → c-Myc signals.

Early studies found critical roles for neutrophils in renal cell carcinoma (RCC) progression. However, detailed mechanisms of how infiltrating neutrophils in the kidney tumor microenvironment impact RCC progression remain unclear. Here we found more neutrophils were infiltrated in human RCC lesions than those found in surrounding normal kidney tissues. Similarly, in vitro studies also revealed that RCC cells recruited more neutrophil HL-60N cells than normal kidney epithelial cells. Furthermore, in vitro and in vivo experiments also showed that the infiltrated neutrophils could promote RCC cell growth. Mechanism studies showed that co-culture of RCC cells with neutrophil HL-60N cells could selectively upregulate the androgen receptor (AR) signals, which might then alter the c-Myc signals. Interruption approaches using AR-siRNA to knock down AR in RCC cells blocked neutrophil-enhanced RCC cell proliferation. In vivo data using an orthotopically xenografted RCC mouse model also confirmed that infiltrated neutrophils could promote RCC proliferation via modulating the expressions of related cytokines. Together, these results conclude that infiltrated neutrophils may function through modulating the AR → c-Myc signals to promote RCC cell proliferation. Targeting this newly identified infiltrating neutrophil → AR → c-Myc signal pathway in the kidney tumor microenvironment may provide a new potential therapy to better suppress RCC progression.