Stromal Signaling Research Articles

Single-cell analysis of mouse and human prostate reveals novel fibroblasts with specialized distribution and microenvironment interactions.

Stromal-epithelial interactions are critical to the morphogenesis, differentiation, and homeostasis of the prostate, but the molecular identity and anatomy of discrete stromal cell types is poorly understood. Using single-cell RNA sequencing, we identified and validated the in situ localization of three smooth muscle subtypes (prostate smooth muscle, pericytes, and vascular smooth muscle) and two novel fibroblast subtypes in human prostate. Peri-epithelial fibroblasts (APOD+) wrap around epithelial structures, whereas interstitial fibroblasts (C7+) are interspersed in extracellular matrix. In contrast, the mouse displayed three fibroblast subtypes with distinct proximal-distal and lobe-specific distribution patterns. Statistical analysis of mouse and human fibroblasts showed transcriptional correlation between mouse prostate (C3+) and urethral (Lgr5+) fibroblasts and the human interstitial fibroblast subtype. Both urethral fibroblasts (Lgr5+) and ductal fibroblasts (Wnt2+) in the mouse contribute to a proximal Wnt/Tgfb signaling niche that is absent in human prostate. Instead, human peri-epithelial fibroblasts express secreted WNT inhibitors SFRPs and DKK1, which could serve as a buffer against stromal WNT ligands by creating a localized signaling niche around individual prostate glands. We also identified proximal-distal fibroblast density differences in human prostate that could amplify stromal signaling around proximal prostate ducts. In human benign prostatic hyperplasia, fibroblast subtypes upregulate critical immunoregulatory pathways and show distinct distributions in stromal and glandular phenotypes. A detailed taxonomy of leukocytes in benign prostatic hyperplasia reveals an influx of myeloid dendritic cells, T cells and B cells, resembling a mucosal inflammatory disorder. A receptor-ligand interaction analysis of all cell types revealed a central role for fibroblasts in growth factor, morphogen, and chemokine signaling to endothelia, epithelia, and leukocytes. These data are foundational to the development of new therapeutic targets in benign prostatic hyperplasia. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stromal induction of BRD4 phosphorylation Results in Chromatin Remodeling and BET inhibitor Resistance in Colorectal Cancer

BRD4, a Bromodomain and Extraterminal (BET) protein family member, is a promising anti-cancer drug target. However, resistance to BET inhibitors targeting BRD4 is common in solid tumors. Here, we show that cancer-associated fibroblast (CAF)-activated stromal signaling, interleukin-6/8-JAK2, induces BRD4 phosphorylation at tyrosine 97/98 in colorectal cancer, resulting in BRD4 stabilization due to interaction with the deubiquitinase UCHL3. BRD4 phosphorylation at tyrosine 97/98 also displays increased binding to chromatin but reduced binding to BET inhibitors, resulting in resistance to BET inhibitors. We further show that BRD4 phosphorylation promotes interaction with STAT3 to induce chromatin remodeling through concurrent binding to enhancers and super-enhancers, supporting a tumor-promoting transcriptional program. Inhibition of IL6/IL8-JAK2 signaling abolishes BRD4 phosphorylation and sensitizes BET inhibitors in vitro and in vivo. Our study reveals a stromal mechanism for BRD4 activation and BET inhibitor resistance, which provides a rationale for developing strategies to treat CRC more effectively.

Macrophage inhibitory cytokine-1 induced by a high-fat diet promotes prostate cancer progression by stimulating tumor-promoting cytokine production from tumor stromal cells.

BackgroundRecent studies have indicated that a high‐fat diet (HFD) and/or HFD‐induced obesity may influence prostate cancer (PCa) progression, but the role of HFD in PCa microenvironment is unclear. This study aimed to delineate the molecular mechanisms of PCa progression under HFD milieus and define the stromal microenvironment focusing on macrophage inhibitory cytokine‐1 (MIC‐1) activation.MethodsWe investigated the effects of HFD on PCa stromal microenvironment and MIC‐1 signaling activation using PC‐3M‐luc‐C6 PCa model mice fed with HFD or control diet. Further, we explored the effect of periprostatic adipocytes derived from primary PCa patients on activation and cytokine secretion of prostate stromal fibroblasts. Expression patterns and roles of MIC‐1 signaling on human PCa stroma activation and progression were also investigated.ResultsHFD stimulated PCa cell growth and invasion as a result of upregulated MIC‐1 signaling and subsequently increased the secretion of interleukin (IL)‐8 and IL‐6 from prostate stromal fibroblasts in PC‐3M‐luc‐C6 PCa mouse model. In addition, periprostatic adipocytes directly stimulated MIC‐1 production from PC‐3 cells and IL‐8 secretion in prostate stromal fibroblasts through the upregulation of adipose lipolysis and free fatty acid release. The increased serum MIC‐1 was significantly correlated with human PCa stroma activation, high serum IL‐8, IL‐6, and lipase activity, advanced PCa progression, and high body mass index of the patients. Glial‐derived neurotrophic factor receptor α‐like (GFRAL), a specific receptor of MIC‐1, was highly expressed in both cytoplasm and membrane of PCa cells and surrounding stromal fibroblasts, and the expression level was decreased by androgen deprivation therapy and chemotherapy.ConclusionHFD‐mediated activation of the PCa stromal microenvironment through metabolically upregulated MIC‐1 signaling by increased available free fatty acids may be a critical mechanism of HFD and/or obesity‐induced PCa progression.

Progression of prostate carcinoma is promoted by adipose stromal cell-secreted CXCL12 signaling in prostate epithelium

Aggressiveness of carcinomas is linked with tumor recruitment of adipose stromal cells (ASC), which is increased in obesity. ASC promote cancer through molecular pathways not fully understood. Here, we demonstrate that epithelial–mesenchymal transition (EMT) in prostate tumors is promoted by obesity and suppressed upon pharmacological ASC depletion in HiMyc mice, a spontaneous genetic model of prostate cancer. CXCL12 expression in tumors was associated with ASC recruitment and localized to stromal cells expressing platelet-derived growth factor receptors Pdgfra and Pdgfrb. The role of this chemokine secreted by stromal cells in cancer progression was further investigated by using tissue-specific knockout models. ASC deletion of CXCL12 gene in the Pdgfr + lineages suppressed tumor growth and EMT, indicating stroma as the key source of CXCL12. Clinical sample analysis revealed that CXCL12 expression by peritumoral adipose stroma is increased in obesity, and that the correlating increase in Pdgfr/CXCL12 expression in the tumor is linked with decreased survival of patients with prostate carcinoma. Our study establishes ASC as the source of CXCL12 driving tumor aggressiveness and outlines an approach to treatment of carcinoma progression.

Protein kinase C-β-dependent changes in the glucose metabolism of bone marrow stromal cells of chronic lymphocytic leukemia

Survival of chronic lymphocytic leukemia (CLL) cells critically depends on the support of an adapted and therefore appropriate tumor microenvironment. Increasing evidence suggests that B-cell receptor-associated kinases such as protein kinase C-β (PKCβ) or Lyn kinase are essential for the formation of a microenvironment supporting leukemic growth. Here, we describe the impact of PKCβ on the glucose metabolism in bone marrow stromal cells (BMSC) upon CLL contact. BMSC get activated by CLL contact expressing stromal PKCβ that diminishes mitochondrial stress and apoptosis in CLL cells by stimulating glucose uptake. In BMSC, the upregulation of PKCβ results in increased mitochondrial depolarization and leads to a metabolic switch toward oxidative phosphorylation. In addition, PKCβ-deficient BMSC regulates the expression of Hnf1 promoting stromal insulin signaling after CLL contact. Our data suggest that targeting PKCβ and the glucose metabolism of the leukemic niche could be a potential therapeutic strategy to overcome stroma-mediated drug resistance.

Abstract SS1-11: Comprehensive analysis of health services, sociodemographic, clinical, and genomic factors driving locally advanced breast cancer mortality via a first-in-kind linkage of SEER-Medicare data with physical tumor samples

Abstract Background: There continues to be an active debate as to the relative contribution of health services, sociodemographic, clinical, and genomic factors in breast cancer disparities. Efforts to understand the contributions of each of these factors have historically focused on separate investigations of socioeconomic, demographic, and genomic elements. However, true multidisciplinary investigation of these factors requires the existence of datasets that combine each of these elements together into population-level, real world cohorts. To this end we created the first-in-kind linkage of SEER-Medicare data to physical tumor samples to combine clinical, health services, and genomic data into a single cohort and used it to investigate the impact of screening and socioeconomic status on early stage breast cancer biology and mortality.Methods: This retrospective study used formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) tissue collected between 1992-2006 within the Iowa and Hawaii SEER Residual Tissue Repositories (SEER-RTR). Medicare fee-for-service claims data were linked for participating patients age 65 and older. Molecular subtyping and exploratory genomic analyses were completed using the NanoString Breast Cancer 360 (BC360) gene expression panel containing the PAM50 classifier. Screening status was assessed by validated claims-based algorithms. Factors associated with overall (OS) & breast cancer-specific (BCS) survival were analyzed in women aged 66-75 with clinically high risk ER+ disease (M0, T1b+, LN+) using Cox proportional hazards models combining sociodemographic, clinical, and genomic data.Results: SEER-Medicare data were available for 1,232 women, of which 379 (30.7%) were diagnosed by screening mammogram. Screen-detected disease was associated with lower T stage, N stage, and improved OS (HR 0.72) & BCS (HR 0.68) in multivariable analysis. Molecular analysis of 130 luminal A/B cases revealed superior outcomes of luminal A and luminal B tumors compared to symptom detected tumors of the same molecular subtype (p = 0.02). In multivariable cox proportional hazards models increased overall mortality was associated with impoverished neighborhoods, PR- receptor status, upregulation of TGF-beta and p53 dysregulation, whereas protective factors included upregulation of androgen receptor (AR), stromal, and cytotoxicity signaling (all P<0.05; Table 1). T2 vs. T1 tumor stage was associated with the largest observed differences in gene expression, and was associated with distinct changes in gene expression in luminal A vs. B disease including down regulation of interferon gamma signaling and MHCII expression in luminal B, but not luminal A, tumors.Conclusions and Relevance: Linkage of SEER, Medicare, and physical tumor specimen data is feasible and allows the combination of genomic analyses with clinical and health services data within population-level cohorts and may provide a novel means to elucidate links between biology, access, and disparities in breast cancer outcomes. Future research is warranted to conduct analogous prospective investigations of cancer disparities. Table – Cox multivariable survival modelParameterAll-cause mortalityHR (95% CI)Symptomatic tumor1.59 (0.70 - 3.64)Stage N2+ vs N12.67 (0.48 -14.83)Stage T2 vs T13.49 (1.42 - 8.58)Aged 71-76 vs 66-702.30 (1.05 - 5.01)Comorbidity Score (ref=0)2+8.85 (1.88-41.73)High school dropout rate3.72 (1.36 -10.17)PR- vs PR+7.25 (1.96 -26.77)Luminal B vs Luminal A0.99 (0.40 - 2.49)AR signature0.21 (0.09 - 0.48)TGF beta signature6.97 (2.00 -24.33)Macrophages signature0.22 (0.08 - 0.60)BC cytotoxicity signature0.61 (0.43 - 0.88)p53 signature3.12 (1.51 - 6.47) Citation Format: Timothy J Robinson, Lauren Wilson, Paul K Marcom, Melissa Troester, Charles F Lynch, Brenda Hernandez, Edgardo P Castellar, Heather Ann Brauer, Lindsey Enewold, Michaela Dinan. Comprehensive analysis of health services, sociodemographic, clinical, and genomic factors driving locally advanced breast cancer mortality via a first-in-kind linkage of SEER-Medicare data with physical tumor samples [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SS1-11.

Tumour microenvironment-based molecular profiling reveals ideal candidates for high-grade serous ovarian cancer immunotherapy.

ObjectiveDue to limited immunological profiles of high‐grade serous ovarian cancer (HGSOC), we aimed to characterize its molecular features to determine whether a specific subset that can respond to immunotherapy exists.Materials and MethodsA training cohort of 418 HGSOC samples from TCGA was analysed by consensus non‐negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGSOCs and in vitro experiments were used for validation.ResultsWe identified immune and non‐immune groups where the former was enriched in signatures that reflect immune cells, infiltration and PD‐1 signalling (all, P < 0.001), and presented with a lower chromosomal aberrations but increased neoantigens, tumour mutation burden, and microsatellite instability (all, P < 0.05); this group was further refined into two microenvironment‐based subtypes characterized by either immunoactivation or carcinoma‐associated fibroblasts (CAFs) and distinct prognosis. CAFs‐immune subtype was enriched for factors that mediate immunosuppression and promote tumour progression, including highly expressed stromal signature, TGF‐β signalling, epithelial‐mesenchymal transition and tumour‐associated M2‐polarized macrophages (all, P < 0.001). Robustness of these immune‐specific subtypes was verified in validation cohorts, and in vitro experiments indicated that activated‐immune subtype may benefit from anti‐PD1 antibody therapy (P < 0.05).ConclusionOur findings revealed two immune subtypes with different responses to immunotherapy and indicated that some HGSOCs may be susceptible to immunotherapies or combination therapies.

Hedgehog Signaling in Colorectal Cancer: All in the Stroma?

Hedgehog (Hh) signaling regulates intestinal development and homeostasis. The role of Hh signaling in cancer has been studied for many years; however, its role in colorectal cancer (CRC) remains controversial. It has become increasingly clear that the “canonical” Hh pathway, in which ligand binding to the receptor PTCH1 initiates a signaling cascade that culminates in the activation of the GLI transcription factors, is mainly organized in a paracrine manner, both in the healthy colon and in CRC. Such canonical Hh signals largely act as tumor suppressors. In addition, stromal Hh signaling has complex immunomodulatory effects in the intestine with a potential impact on carcinogenesis. In contrast, non-canonical Hh activation may have tumor-promoting roles in a subset of CRC tumor cells. In this review, we attempt to summarize the current knowledge of the Hh pathway in CRC, with a focus on the tumor-suppressive role of canonical Hh signaling in the stroma. Despite discouraging results from clinical trials using Hh inhibitors in CRC and other solid cancers, we argue that a more granular understanding of Hh signaling might allow the exploitation of this key morphogenic pathway for cancer therapy in the future.

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

A Wnt-Induced Phenotypic Switch in Cancer-Associated Fibroblasts Inhibits EMT in Colorectal Cancer.

Tumor progression is recognized as a result of an evolving cross-talk between tumor cells and their surrounding nontransformed stroma. Although Wnt signaling has been intensively studied in colorectal cancer, it remains unclear whether activity in the tumor-associated stroma contributes to malignancy. To specifically interfere with stromal signals, we generated Wnt-independent tumor organoids that secrete the Wnt antagonist Sfrp1. Subcutaneous transplantation into immunocompetent as well as immunodeficient mice resulted in a strong reduction of tumor growth. Histologic and transcriptomic analyses revealed that Sfrp1 induced an epithelial-mesenchymal transition (EMT) phenotype in tumor cells without affecting tumor-intrinsic Wnt signaling, suggesting involvement of nonimmune stromal cells. Blockage of canonical signaling using Sfrp1, Dkk1, or fibroblast-specific genetic ablation of β-catenin strongly decreased the number of cancer-associated myofibroblasts (myCAF). Wnt activity in CAFs was linked with distinct subtypes, where low and high levels induced an inflammatory-like CAF (iCAF) subtype or contractile myCAFs, respectively. Coculture of tumor organoids with iCAFs resulted in significant upregulation of EMT markers, while myCAFs reverted this phenotype. In summary, we show that tumor growth and malignancy are differentially regulated via distinct fibroblast subtypes under the influence of juxtacrine Wnt signals. SIGNIFICANCE: This study provides evidence for Wnt-induced functional diversity of colorectal cancer-associated fibroblasts, representing a non-cell autonomous mechanism for colon cancer progression. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5569/F1.large.jpg.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis

Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor β and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Abstract PO-049: Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is resistant to immune checkpoint blockade due to its intrinsic immunosuppressive properties. The fibroinflammatory stroma, particularly inflammatory cancer associated fibroblasts (CAFs), are a key determinant of the immune microenvironment in PDAC, however therapies aimed at broad-based targeting of PDAC stroma have been decidedly unsuccessful. Recently, interleukin-1α (IL-1) has emerged as a critical mediator of CAF activation and orchestrator of immune suppression in PDAC. We sought to explore the effects of IL-1 blockade on reprogramming the immune microenvironment and enhancing susceptibility to checkpoint inhibition through suppression of inflammatory stromal signaling in PDAC. Methods: Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the recombinant IL-1 receptor antagonist (anakinra, 50 mg/kg IP BID) or vehicle control. Single-cell cDNA library generation was performed using the 10× Chromium System. Expression matrices were processed using the R package Seurat 3.0 and clustered using AUC-based scoring algorithm. Immunophenotyping experiments were performed on single-cell suspensions using the Cytoflex platform. For survival studies, PKT mice were treated with vehicle, anti-PD1 antibody (200 μg/twice weekly), anakinra, or combination until moribund. Tissues were further processed for downstream qPCR and histologic analyses. Human pancreatic stellate cell (hPSC) and immortalized CAF cell lines procured from patient-derived xenografts were utilized for in vitro mechanistic studies. Results: Single-cell analysis revealed that anakinra treatment in PKT mice reduced levels of numerous pro-inflammatory CAF cytokines, including Cxcl1 and Il6, within CAF subclusters. Confirmatory qPCR of bulk tumor samples revealed a significant reduction in Cxcl1 and Il6 gene transcription and a global reduction in stromal fibrosis with anakinra treatment. IL-1 inhibition selectively reduced alternatively-activated macrophage (CD11b+F480+CD206+) and PMN-MDSC (CD11b+Ly6G+) populations within PDAC tumors. Combination treatment of anakinra with anti-PD1 antibody reduced tumor weight, increased intratumoral levels of effector memory (CD8+CD44+CD62L-) and activated cytotoxic (CD8+CD107+) T cells, and improved overall survival in PKT mice. In vitro studies demonstrated that conditioned media from PDAC tumor cells significantly activated Cxcl1 and Il6 gene transcription in hPSCs and CAFs in an IL-1α-dependent manner. IL-1-mediated transcription of these cytokines was abrogated by selective inhibition of both the p38 MAPK and NFκB cascades, demonstrating a coordinated effort between these signaling pathways in modulating the IL-1-induced stress response in stromal cells. Conclusions: These findings provide important mechanistic data and compelling pre-clinical evidence to explore IL-1 inhibition in combination with immune checkpoint blockade in PDAC patients. Citation Format: Austin R. Dosch, Samara Singh, Xizi Dai, Siddharth Mehra, Anna Bianchi, Iago De Castro Silva, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-049.

Uterine Epithelial Progesterone Receptor Governs Uterine Receptivity Through Epithelial Cell Differentiation.

Progesterone receptor (PGR) is indispensable for pregnancy in mammals. Uterine PGR responds to the heightened levels of ovarian progesterone (P4) after ovulation and regulates uterine gene transcription for successful embryo implantation. Although epithelial and stromal P4-PGR signaling may interact with each other to form appropriate endometrial milieu for uterine receptivity and the subsequent embryo attachment, it remains unclear what the specific roles of epithelial P4-PGR signaling in the adult uterus are. Here we generated mice with epithelial deletion of Pgr in the adult uterus (Pgrfl/flLtfCre/+ mice) by crossing Pgr-floxed and Ltf-Cre mice. Pgrfl/flLtfCre/+ mice are infertile due to the impairment of embryo attachment. Pgrfl/flLtfCre/+ uteri did not exhibit epithelial growth arrest, suggesting compromised uterine receptivity. Both epithelial and stromal expressions of P4-responsive genes decreased in Pgrfl/flLtfCre/+ mice during the peri-implantation period, indicating that epithelial Pgr deletion affects not only epithelial but stromal P4 responsiveness. In addition, uterine LIF, an inducer of embryo attachment, was decreased in Pgrfl/flLtfCre/+ mice. The RNA-seq analysis using luminal epithelial specimens dissected out by laser capture microdissection revealed that the signaling pathways related to extracellular matrix, cell adhesion, and cell proliferation are altered in Pgr fl/flLtf Cre/+ mice. These findings suggest that epithelial PGR controls both epithelial and stromal P4 responsiveness and epithelial cell differentiation, which provides normal uterine receptivity and subsequent embryo attachment.

Abstract B15: Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome

Abstract In this study, we are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni syndrome (LFS). LFS is a cancer predisposition syndrome that is associated with germline mutations in TP53. Individuals with LFS are faced with a significant lifetime risk of developing a wide spectrum of early-onset malignancies, thus necessitating the identification of pharmacologic agents to prevent tumor onset. Inhibitors of the mTORC1 pathway, namely rapamycin and metformin, have been shown to promote lifespan extension and delay or even prevent tumor onset in LFS mouse models. These findings are unsurprising—since wild-type p53 inhibits mTORC1—yet noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS. mTORC1 signaling has been implicated in promoting the protumorigenic senescence-associated secretory phenotype (SASP). The SASP constitutes the release of soluble factors, such as cytokines, growth factors, and proteases from senescent cells into the microenvironment, thus promoting oncogenesis in a paracrine manner. Interestingly, an increased incidence of senescence has been reported in LFS patients, and mutant p53 has been shown to promote the secretion of a quantitatively and qualitatively more protumorigenic SASP. As such, we believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of protumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are 1) to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and 2) to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes. To address these objectives, we co-injected patient-derived LFS fibroblasts with tumor cells of a common LFS subtype (rhabdomyosarcoma [RMS]) into immunocompromised mice and observed that tumors formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumors formed from co-injection with non-LFS fibroblasts. To determine whether LFS fibroblasts secrete more protumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures and saw that LFS fibroblasts secrete markedly higher levels of protumorigenic, SASP-related cytokines. To investigate the protumorigenic effects of fibroblast secreted factors, we treated spheroids formed from tumor cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust proproliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth. Thus far, we have observed that LFS stromal constituents favor tumor cell proliferation in vitro and in vivo, and based on the current literature, we believe that mTORC1 signaling plays a role in favoring these processes. Citation Format: Camilla M. Giovino, Nish Patel, Sangeetha Paramathas, David Malkin, Ran Kafri. Investigating the contribution of mTORC1-dependent stromal signaling to cancer onset in Li-Fraumeni syndrome [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B15.

Abstract 3977: Stromal BMP signaling imbalance mediated by GREM1 and ISLR regulates colorectal cancer progression

Abstract Introduction Cancer-associated fibroblasts (CAFs), a heterogeneous component of the tumor microenvironment, substantially influence tumor progression. Bone morphogenetic proteins (BMP) play a critical part in defining the intestinal epithelial niche and either promote or retard cancer progression in a context-dependent manner. However, the role of BMP signaling in the colorectal cancer (CRC) stroma remains to be fully elucidated. This study investigated the significance of mesenchymal BMP signaling as a potential therapeutic target in CRC progression. Design Using CRC expression array data, we identified two CAF-specific factors involved in BMP signaling, then verified their upregulation in the human CRC stroma by in-situ hybridization (ISH). We took advantage of a preclinical mouse model of CRC hepatic metastasis to test approaches targeting the BMP signaling pathway. Results CRC microarray data identified GREM1 and ISLR as CAF-specific genes involved in BMP signaling. In colonic myofibroblasts, Grem1-overexpression inhibited BMP signaling whereas BMP7 signaling was augmented by Islr overexpression, suggesting opposing roles for GREM1 and ISLR in the regulation of BMP signaling. ISH using human rectal cancer samples revealed that GREM1 and ISLR were expressed in distinct CAF subpopulations and that GREM1 and ISLR expression predicted poor and favorable survival, respectively. Notably, Grem1 and Islr expression was differentially regulated by Foxl1, an intestinal mesenchyme-lineage transcription factor, and TGF-b, indicating a mechanism for generating fibroblast heterogeneity. Finally, adeno-associated virus 8-mediated in-vivo overexpression of Islr in hepatocytes retarded growth and generated more differentiated histology in CRC hepatic metastases. Conclusion These data suggest that increased stromal BMP signaling may ameliorate CRC progression and provide a rationale for targeting stromal BMP signaling to inhibit CRC progression and metastasis. Citation Format: Hiroki Kobayashi, Krystyna A. Gieniec, Tongtong Wang, Josephine A. Wright, Nobumi Suzuki, Tamsin RM Lannagan, Yoku Hayakawa, Simon J. Leedham, Nicholas Arpaia, Siddhartha Mukherjee, Timothy C. Wang, Atsushi Enomoto, Masahide Takahashi, Daniel L. Worthley, Susan L. Woods. Stromal BMP signaling imbalance mediated by GREM1 and ISLR regulates colorectal cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3977.

Abstract 3985: GDF15 expression level impacts the canonical pathways of the tumor microenvironment and ovarian tumors with wild-type p53 in response to chemotherapy

Abstract Background The standard treatment for ovarian cancer is surgery, followed by chemotherapy with a platinum agent, such as cisplatin or carboplatin, and a taxane, such as paclitaxel. We previously observed that patients with ovarian cancer that was wild-type for p53 had a lower survival rate than did those with p53 mutations. A better understanding of the molecular changes that occur in epithelial ovarian cancer cells with wild-type p53 in response to cisplatin treatment could reveal a novel mechanism of chemoresistance. Therefore, we might be able to develop novel strategy to overcome chemoresistance. Methods Gene expression profiling was performed on the wild-type p53 ovarian cancer cell line A2780 to identify genes that are highly induced by cisplatin. Growth differentiation factor 15 (GDF15) was identified as being highly induced by cisplatin or carboplatin. This finding was validated in a panel of wild-type and mutant p53 ovarian cancer cell lines. Mouse orthotopic models were used to further investigate the induction of GDF15 by cisplatin in vivo and the effect of GDF15 knockdown (GDF15-KD) on tumor growth and cisplatin response. A novel analysis was performed of RNAseq data generated from tumors formed in cisplatin-treated orthotopic mouse models to understand the tumor and stromal cell signaling changes. Results The induction of GDF15 was demonstrated in vivo, and the serum level of GDF15 after cisplatin treatment was also found to be significantly up-regulated in an A2780 ovarian cancer orthotopic mouse model. More importantly, cisplatin treatment was more effective in shrinking the mouse tumors developed from GDF15-KD A2780 cell line than did control tumors. Histopathological and novel RNAseq analyses of mouse tumors revealed that GDF15-KD tumors had a lower stromal component than did control tumors after cisplatin treatment. GDF15-KD tumors also had a different repertoire of activated and inhibited canonical pathways in their stromal cell and cancer cell components than did control tumors in response to cisplatin treatment. Conclusions GDF15 expression in wild-type p53 ovarian cancer cells can modulate the canonical pathways in the tumor microenvironment in response to cisplatin and may be a mechanism of chemoresistance. Targeting GDF15 signaling could be a novel strategy to enhance the effectiveness of chemotherapy. Citation Format: Daisy I. Izaguirre, Yvonne TM Tsang, Eucharist H. Kun, David M. Gershenson, Kwong-Kwok Wong. GDF15 expression level impacts the canonical pathways of the tumor microenvironment and ovarian tumors with wild-type p53 in response to chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3985.

Stromal CCL2 Signaling Promotes Mammary Tumor Fibrosis through Recruitment of Myeloid-Lineage Cells.

Obesity is correlated with breast tumor desmoplasia, leading to diminished chemotherapy response and disease-free survival. Obesity causes chronic, macrophage-driven inflammation within breast tissue, initiated by chemokine ligand 2 (CCL2) signaling from adipose stromal cells. To understand how CCL2-induced inflammation alters breast tumor pathology, we transplanted oncogenically transformed human breast epithelial cells with breast stromal cells expressing CCL2 or empty vector into murine mammary glands and examined tumor formation and progression with time. As tumors developed, macrophages were rapidly recruited, followed by the emergence of cancer-associated fibroblasts (CAFs) and collagen deposition. Depletion of CD11b + myeloid lineage cells early in tumor formation reduced tumor growth, CAF numbers, and collagen deposition. CCL2 expression within developing tumors also enhanced recruitment of myeloid progenitor cells from the bone marrow into the tumor site. The myeloid progenitor cell population contained elevated numbers of fibrocytes, which exhibited platelet-derived growth factor receptor-alpha (PDGFRα)-dependent colony formation and growth in vitro. Together, these results suggest that chronic inflammation induced by CCL2 significantly enhances tumor growth and promotes the formation of a desmoplastic stroma through early recruitment of macrophages and fibrocytes into the tumor microenvironment. Fibrocytes may be a novel target in the tumor microenvironment to reduce tumor fibrosis and enhance treatment responses for obese breast cancer patients.

Carcinoma-Associated Mesenchymal Stem Cells Promote Chemoresistance in Ovarian Cancer Stem Cells via PDGF Signaling.

Within the ovarian cancer tumor microenvironment, cancer stem-like cells (CSC) interact with carcinoma associated mesenchymal stem/stromal cells (CA-MSC) through multiple secreted cytokines and growth factors. These paracrine interactions have been revealed to cause enrichment of CSC and their chemoprotection; however, it is still not known if platelet-derived growth factor (PDGF) signaling is involved in facilitating these responses. In order to probe this undiscovered bidirectional communication, we created a model of ovarian malignant ascites in the three-dimensional (3D) hanging drop heterospheroid array, with CSC and CA-MSC. We hypothesized that PDGF secretion by CA-MSC increases self-renewal, migration, epithelial to mesenchymal transition (EMT) and chemoresistance in ovarian CSC. Our results indicate that PDGF signaling in the CSC-MSC heterospheroids significantly increased stemness, metastatic potential and chemoresistance of CSC. Knockdown of PDGFB in MSC resulted in abrogation of these phenotypes in the heterospheroids. Our studies also reveal a cross-talk between PDGF and Hedgehog signaling in ovarian cancer. Overall, our data suggest that when the stromal signaling via PDGF to ovarian CSC is blocked in addition to chemotherapy pressure, the tumor cells are significantly more sensitive to chemotherapy. Our results emphasize the importance of disrupting the signals from the microenvironment to the tumor cells, in order to improve response rates. These findings may lead to the development of combination therapies targeting stromal signaling (such as PDGF and Hedgehog) that can abrogate the tumorigenic, metastatic and platinum resistant phenotypes of ovarian CSC through additional investigations.

Tumor-Associated Macrophages in Osteosarcoma: From Mechanisms to Therapy.

Osteosarcomas (OSs) are bone tumors most commonly found in pediatric and adolescent patients characterized by high risk of metastatic progression and recurrence after therapy. Effective therapeutic management of this disease still remains elusive as evidenced by poor patient survival rates. To achieve a more effective therapeutic management regimen, and hence patient survival, there is a need to identify more focused targeted therapies for OSs treatment in the clinical setting. The role of the OS tumor stroma microenvironment plays a significant part in the development and dissemination of this disease. Important components, and hence potential targets for treatment, are the tumor-infiltrating macrophages that are known to orchestrate many aspects of OS stromal signaling and disease progression. In particular, increased infiltration of M2-like tumor-associated macrophages (TAMs) has been associated with OS metastasis and poor patient prognosis despite currently used aggressive therapies regimens. This review aims to provide a summary update of current macrophage-centered knowledge and to discuss the possible roles that macrophages play in the process of OS metastasis development focusing on the potential influence of stromal cross-talk signaling between TAMs, cancer-stem cells and additional OSs tumoral microenvironment factors.

Dual role of IL6 mediated by mesenchymal stromal cell signaling to joint macrophages in osteoarthritis

Purpose: Osteoarthritis (OA) is a joint disease affecting > 5 million Canadians and patients have limited palliative and joint-replacement surgical options. Stromal cell therapy is emerging as a compelling treatment for OA. Our group led the first-in-Canada Ph1/2 trial with bone marrow mesenchymal stromal cells (BM-MSCs) in OA patients successfully; and significant improvements in patient outcomes were observed. This was accompanied with reduction in pro-inflammatory monocytes/macrophages (MΦs) in the synovial fluid (SF), suggestive of a clinical MSC anti-inflammatory effect. Understanding mechanism of action of MSCs in OA will enable effective donor MSC screening, and generation of more efficient engineered MSCs. While IL6 is a known pro-inflammatory mediator in the joint, and anti-IL6 therapeutics have been effective in treatment of Rheumatoid Arthritis, blocking single pro-inflammatory cytokines has been less effective in OA. This leads us to believe that IL6 might have complex roles. Indeed, MSCs-induced polarization of macrophages to inflammation resolving subtypes via IL6 signaling. This effect is not well studied in an OA joint where high levels of IL6 may have pro-inflammatory effects. We hypothesize that IL-6, which is known as pro-inflammatory mediator in OA is mediating anti-inflammatory function of MSCs through signaling via membrane-bound, but not soluble receptors. Methods: We co-cultured peripheral blood-derived MΦs with OA BM-MSCs in the presence of early and late SF from OA patients; or in the presence of IL6 antibody. Polarization of MΦs were assessed by analysis of surface markers and IL10 secretion. Levels of IL6 soluble IL6 receptor (sIL6R) were measured by ELISA. Results: Preliminary quantification of BM- MSC from OA patients revealed high IL6 secretion is associated with positive patient-reported outcomes. IL6 antibody abrogated MSCs-induced anti-inflammatory polarization of MΦs as shown by reduction of CD163 and CD206, suggesting a key role for IL6 in MSC anti-inflammatory effects on MΦs. Paradoxically, IL6 levels were increased in SF from late stage OA, whereas levels of sIL6R were comparable in early and late OA-SF. Further, the effect of MSCs on anti-inflammatory polarization of MΦs was attenuated in the presence of late OA-SF compared to early OA-SF. This could be due to enhanced IL6 trans-signaling (mediated by sIL6R) in late-stage OA SF expected to occur at the expense of reduced IL6 classical pathway (mediated by membrane bound IL6 receptor, mIL6R). To discriminate between classical vs. trans-signalling, we are using soluble gp130, a non-signaling decoy receptor which complexes IL6-sIL6R. Our preliminary result showed that sgp130 treatment enhanced the expression of CD163 in MΦs exposed to pooled OA SF. The classical IL6 pathway will be studied specifically by silencing mIL6R in MΦs using siRNA Conclusions: MSCs exert their anti-inflammatory action in OA, at least partly through polarization of MΦs to anti-inflammatory phenotype mediated by IL6. The ratio of IL6 signaling through trans-vs. classical signaling pathways is an important arbiter of MSC-modulation of joint inflammation.