Stromal Signaling Research Articles

Secreted stromal protein ISLR promotes intestinal regeneration by suppressing epithelial Hippo signaling.

The Hippo-YAP signaling pathway plays an essential role in epithelial cells during intestinal regeneration and tumorigenesis. However, the molecular mechanism linking stromal signals to YAP-mediated intestinal regeneration and tumorigenesis is poorly defined. Here, we report a stroma-epithelium ISLR-YAP signaling axis essential for stromal cells to modulate epithelial cell growth during intestinal regeneration and tumorigenesis. Specifically, upon inflammation and in cancer, an oncogenic transcription factor ETS1 in stromal cells induces expression of a secreted protein ISLR that can inhibit Hippo signaling and activate YAP in epithelial cells. Deletion of Islr in stromal cells in mice markedly impaired intestinal regeneration and suppressed tumorigenesis in the colon. Moreover, the expression of stromal cell-specific ISLR and ETS1 significantly increased in inflamed mucosa of human IBD patients and in human colorectal adenocarcinoma, accounting for the epithelial YAP hyperactivation. Collectively, our findings provide new insights into the signaling crosstalk between stroma and epithelium during tissue regeneration and tumorigenesis.

TSLP and IL-33 reciprocally promote each other's lung protein expression and ILC2 receptor expression to enhance innate type-2 airway inflammation.

BackgroundThe epithelial cell‐derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL‐33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL‐33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.MethodsBALB/c WT and TSLP receptor‐deficient (TSLPR−/−) mice were challenged intranasally with Alternaria extract (Alt‐Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre‐administered with rTSLP or vehicle, TSLPR−/− mice, and IL‐33 receptor‐deficient (ST2−/−) mice were challenged intranasally with Alt‐Ext or vehicle once or twice to evaluate IL‐33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL‐33, rTSLP + rIL‐33, or vehicle.ResultsThymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL‐5 and IL‐13 following Alt‐Ext‐challenge compared to WT mice. Further, eosinophilia, protein level of lung IL‐4, IL‐5, and IL‐13, and airway mucus score were also significantly decreased in TSLPR−/− mice compared to WT mice. Endogenous and exogenous TSLP increased Alt‐Ext‐induced IL‐33 release into BALF, and ST2 deficiency decreased Alt‐Ext‐induced TSLP expression in the lung. Further, rTSLP and rIL‐33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.ConclusionThymic stromal lymphopoietin and IL‐33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt‐Ext‐challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.

Abstract P6-06-08: Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment

Abstract Purpose: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by visible skin symptoms on the breast at the time of presentation. We demonstrated mesenchymal stem cells (MSC) promote skin symptoms in the SUM149 xenograft model. Here, we extend this finding to IBC patient-derived xenograft (PDX) lines and performed RNA-sequencing analysis of tumors from IBC patient-derived xenograft (PDX) lines by presence of skin symptoms. We hypothesize host MSC variation may explain sporadic skin symptoms in IBC PDX tumors across generations. Methods: MSC were co-injected at the time of tumor initiation in PDX tumors (IBC-3, 5, 6, and 7) and skin symptoms assessed visually and at the time of gross resection. A priori analysis plan was to group PDX by treatment group irrespective of PDX line. IBC PDX models spontaneously but inconsistently develop skin symptoms after multiple passages. RNA sequencing was performed on IBC-PDX tumors (n=33) and non-IBC PDX tumors (n=12) across generations from IBC PDX lines 5, 6, and 7 and non-IBC PDX lines 2, 3, and 4. Four samples were discarded due to poor quality. TSNE was applied using the top differential expressed genes. Xenome analysis was performed to examine host stromal expression by skin involvement. Samples from PDX lines 5 were compared with lines 6 and 7 and the top differentially expressed genes were compared with the Molecular Signatures Databases (MSigDB). CIBERSORT was performed to deconvolute cell type composition. Results: Co-injecting MSC with PDX transplant increased skin symptoms (57% of PDX animals (cohort from IBC5, IBC6, IBC7 and non-IBC4) with MSC vs. 0% without (cohort from IBC5, IBC6, IBC7, non-IBC2 and non-IBC4) (P = 0.0007). TSNE analysis of IBC PDX split samples into three subgroups distinguished by PDX lines and skin invasion. Xenome was used to split reads from human or mouse: samples show high percentage of reads coming from human (mean±SD, 97.44%± 8.98%). Seventy-four genes show FDR ≤ 0.2 between PDX line 5 and line 6 and comparing these top 74 genes with MSigDb for interaction revealed pathways such as GPCR, signal transduction, and sensory perception. CIBERSORT revealed no enrichment for MSC in IBC PDX with skin invasion, however significant differences in CD8 expression in these immunosuppressed PDX models casts doubt on the robustness of this finding. Conclusions: Exogenous MSC enhance skin symptoms in a cohort of IBC and non-IBC PDX. Skin symptoms segregate IBC PDX tumors by expression suggesting specific biology drives this presentation. However, we are unable to demonstrate evidence for spontaneous MSC signaling in these models. Future work is warranted to link top skin symptom related signaling to stromal signals and unravel the unexpected CD8 signaling identified in these models. Citation Format: Omar Rahal, Jie Yang, Li Li, Richard Larson, Steven Van Laere, Naoto Ueno, Erik Sulman, Wendy Woodward. Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-08.

Increased stiffness of the tumor microenvironment in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling

Colorectal cancer (CRC) is the second deadliest cancer in the US due to its propensity to metastasize. Stromal cells and especially cancer-associated fibroblasts (CAF) play a critical biophysical role in cancer progression, but the precise pro-metastatic mechanisms are not clear. Activin A, a TGF-β family member, is a strong pro-metastatic cytokine in the context of CRC. Here, we assessed the link between biophysical forces and pro-metastatic signaling by testing the hypothesis that CAF-generated mechanical forces lead to activin A release and associated downstream effects. Consistent with our hypothesis, we first determined that stromal activin A secretion increased with increasing substrate stiffness. Then we found that stromally-secreted activin A induced ligand-dependent CRC epithelial cell migration and epithelial to mesenchymal transition (EMT). In addition, serum activin A levels are significantly increased in metastatic (stage IV) CRC patients (1.558 ng/ml versus 0.4179 ng/ml, p < 0.05). We propose that increased tumor microenvironment stiffness leads to stromal cell-mediated TGF-β family signaling relying on the induction and utilization of activin A signaling.

Pancreatic Tumor Microenvironment.

The pancreatic ductal adenocarcinoma (PDAC) microenvironment is a diverse and complex milieu of immune, stromal, and tumor cells and is characterized by a dense stroma, which mediates the interaction between the tumor and the immune system within the tumor microenvironment (TME). The interaction between stromal and tumor cells signals and shapes the immune infiltration of TME. The desmoplastic compartment contains infiltrated immune cells including tumor-associated macrophages (TAMs) and large numbers of fibroblasts/myofibroblasts dominated by pancreatic stellate cells (PSCs) which contribute to fibrosis. The highly fibrotic stroma with its extensive infiltration of immunosuppressive cells forms the major component of the pro-tumorigenic microenvironment (Laklai et al. Nat Med 22:497-505, 2016, Zhu et al. Cancer Res 74:5057-5069, 2014) provides a barrier to the delivery of cytotoxic agents and limits T-cell access to tumor cells (Feig et al. Proc Natl Acad Sci USA 110:20212-20217, 2013, Provenzano et al Cancer Cell 21:418-429, 2012). Activated PSCs reduced infiltration of cytotoxic T cells to the juxtatumoral stroma (immediately adjacent to the tumor epithelial cells) of PDAC (Ene-Obong et al. Gastroenterology 145:1121-1132, 2013). M1 macrophages activate an immune response against tumor, but M2 macrophages are involved in immunosuppression promoting tumor progression (Noy and Pollard Immunity 41:49-61, 2014, Ruffell et al. Trends Immunol 33:119-126, 2012). The desmoplastic stroma is reported to protect tumor cells against chemotherapies, promoting their proliferation and migration. However, experimental depletion of the desmoplastic stroma has led to more aggressive cancers in animal studies (Nielsen et al. World J Gastroenterol 22:2678-2700, 2016). Hence reprogramming rather than simple depletion of the PDAC stroma has the potential for developing new therapeutic strategies for PC treatment. Modulation of PSCs/fibrosis and immune infiltration/inflammation composes the major aspects of TME reprogramming.

Androgen action in cell fate and communication during prostate development at single-cell resolution.

Androgens/androgen receptor (AR)-mediated signaling pathways are essential for prostate development, morphogenesis and regeneration. Specifically, stromal AR signaling has been shown to be essential for prostatic initiation. However, the molecular mechanisms underlying AR-initiated mesenchymal-epithelial interactions in prostate development remain unclear. Here, using a newly generated mouse model, we have directly addressed the fate and role of genetically marked AR-expressing cells during embryonic prostate development. Androgen signaling-initiated signaling pathways were identified in mesenchymal niche populations at single-cell transcriptomic resolution. The dynamic cell-signaling networks regulated by stromal AR were additionally characterized in relation to prostatic epithelial bud formation. Pseudotime analyses further revealed the differentiation trajectory and fate of AR-expressing cells in both prostatic mesenchymal and epithelial cell populations. Specifically, the cellular properties of Zeb1-expressing progenitors were assessed. Selective deletion of AR signaling in a subpopulation of mesenchymal rather than epithelial cells dysregulated the expression of the master regulators and significantly impaired prostatic bud formation. These data provide novel, high-resolution evidence demonstrating the important role of mesenchymal androgen signaling in the cellular niche controlling prostate early development by initiating dynamic mesenchyme-epithelia cell interactions.

LAM-003, a new drug for treatment of tyrosine kinase inhibitor–resistant FLT3-ITD–positive AML

AbstractAcute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor–resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.

IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy.

Tumor-derived cytokines are known to drive the catabolism of host tissues, including skeletal muscle. However, our understanding of the specific cytokines that initiate this process remains incomplete. In the current study, we conducted multiplex analyte profiling of cytokines in conditioned medium (CM) collected from human pancreatic cancer (PC) cells, human tumor-associated stromal (TAS) cells, and their co-culture. Of the factors identified, interleukin-8 (IL-8) is released at high levels from PC cells and PC/TAS co-culture and has previously been associated with low muscle mass in cancer patients. We, therefore, treated C2C12 myotubes with IL-8 which led to the activation of ERK1/2, STAT, and Smad signaling, and induced myotube atrophy. Moreover, the treatment of mice with IL-8 also induced significant muscle wasting, confirming the in vivo relevance of IL-8 on muscle. Mechanistically, IL-8-induced myotube atrophy is inhibited by treatment with the CXCR2 antagonist, SB225002, or by treatment with the ERK1/2 inhibitor, U0126. We further demonstrate that this axis mediates muscle atrophy induced by pancreatic cancer cell CM, as neutralization of IL-8 or treatment with SB225002 or U0126 significantly inhibit CM-induced myotube atrophy. Thus, these data support a key role of IL-8 released from human PC cells in initiating atrophy of muscle cells via CXCR2-ERK1/2.

Abstract TMIM-079: INHIBITION OF TUMOR MICROENVIRONMENT CYTOKINE SIGNALING SENSITIZES OVARIAN CANCER CELLS TO ANTIESTROGEN THERAPY

Abstract OBJECTIVE: Ovarian cancer tumor cell estrogen receptor (ER) expression alone is a poor predictor of response to antiestrogen therapy and some patients with ER-negative cancers still demonstrate clinical response to therapy, suggesting modifiers of response that can potentially be targeted to improve outcomes. We have demonstrated that IL6 and LIF cytokine signaling from carcinoma-associated mesenchymal stem cells (CA-MSC) in the tumor microenvironment promotes tumorigenesis and hypothesize that this cytokine signaling also engages in crosstalk with estrogen signaling pathways. Furthermore, we propose that blocking both cytokine and estrogen signaling will improve anti-cancer effects. METHODS: High grade serous ovarian cancer (HGSOC) cell lines were treated with CA-MSC conditioned media or recombinant cytokines IL6 and/or LIF. Ovarian cancer cells were then assessed for (i) activation of estrogen response element (ERE)-driven luciferase reporter constructs (SABiosciences), (ii) changes in levels of reported ER target genes by qRT-PCR, and (iii) ER-alpha expression levels by immunoblotting. ER-alpha expression in CA-MSC was assessed by immunoblotting. We then assessed the effect of treating cells with ruxolitinib, the FDA-approved inhibitor of the Janus-associated kinase (JAK) protein that is downstream of IL6/LIF signaling. Tumor cells in the absence or presence of CA-MSC conditioned media were treated with ruxolitinib without or with anti-estrogen therapy. Anti-estrogens studied include the selective estrogen receptor modulator tamoxifen, the selective estrogen receptor downregulator fulvestrant, and the aromatase inhibitor letrozole. Following drug treatment, cell viability was assessed with the MTT assay and colony forming assays, and signaling cascade protein expression determined by immunoblotting. Synergy was calculated by the Chou-Talalay method using CompuSyn software. RESULTS: IL6 and LIF induce ERE reporter construct activation in HGSOC cell lines to a similar extent as control estradiol treatment and increase the expression of known ER target genes. Additionally, IL6 and LIF increase ER-alpha levels in HGSOC cell lines as detected by immunoblotting. The treatment of HGSOC cells with both ruxolitinib and antiestrogen therapy results in a synergistic decrease in cell viability. Variable effects are noted depending on the specific antiestrogen used and the cell line studied, indicating subtle mechanistic differences between cell lines. CA-MSC express ER-alpha, suggesting antiestrogens may exert their effects at least in part through changes in stromal signaling. Further studies are underway, including characterization of effects in both primary tumor cells and animal models. CONCLUSIONS: IL6 and LIF signaling from the tumor microenvironment promotes ovarian cancer cell estrogen signaling. The combination of inhibiting IL6/LIF signaling with ruxolitinib and antiestrogen therapy results in a synergistic decrease in ovarian cancer tumor cell viability. Given the clinical tolerability of antiestrogen therapy with a low side effect profile, strategies such as ruxolitinib treatment to sensitize tumor cells to antiestrogens are an exciting area that warrant further study. Citation Format: Lijun Tan, Victoria Prince, Jake Erba, Karen McLean. INHIBITION OF TUMOR MICROENVIRONMENT CYTOKINE SIGNALING SENSITIZES OVARIAN CANCER CELLS TO ANTIESTROGEN THERAPY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-079.

Tumour Microenvironment-Based Molecular Profiling Reveals Ideal Candidates for High-Grade Serous Ovarian Cancer Immunotherapy

Background: Due to limited immunological profiles of high-grade serous ovarian cancer (HGS-OvCa), we aimed to characterise its molecular features to determine whether a specific subset that can respond to immunotherapy exists. Methods: A training cohort of 418 HGS-OvCa 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 HGS-OvCa samples and in vitro experiments were used for validation. Findings: We identified immune and normal 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 burden of chromosomal aberrations but significantly 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. The 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). The robustness of these immune-specific subtypes were verified in validation cohorts, and in vitro experiments indicated the activated-immune subtype can benefit from anti-PD1 antibody therapy (P<0·05). Interpretation: Our findings revealed two distinct immune subtypes with different responses to immunotherapy and indicate some HGS-OvCas may be susceptible to immunotherapies or combination therapies. Funding Statement: This work was supported by the Fundamental Research Funds for the Central Universities grants of China (Grant No. 2632018FY04); the “Double First-Class” University project (CPU2018GY09); the National Natural Science Foundation of China (81973145). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Not required.

Epigenetic changes in fibroblasts drive cancer metabolism and differentiation.

Genomic changes that drive cancer initiation and progression contribute to the co-evolution of the adjacent stroma. The nature of the stromal reprogramming involves differential DNA methylation patterns and levels that change in response to the tumor and systemic therapeutic intervention. Epigenetic reprogramming in carcinoma-associated fibroblasts are robust biomarkers for cancer progression and have a transcriptional impact that support cancer epithelial progression in a paracrine manner. For prostate cancer, promoter hypermethylation and silencing of the RasGAP, RASAL3 that resulted in the activation of Ras signaling in carcinoma-associated fibroblasts. Stromal Ras activity initiated a process of macropinocytosis that provided prostate cancer epithelia with abundant glutamine for metabolic conversion to fuel its proliferation and a signal to transdifferentiate into a neuroendocrine phenotype. This epigenetic oncogenic metabolic/signaling axis seemed to be further potentiated by androgen receptor signaling antagonists and contributed to therapeutic resistance. Intervention of stromal signaling may complement conventional therapies targeting the cancer cell.

Sufu- and Spop-mediated downregulation of Hedgehog signaling promotes beta cell differentiation through organ-specific niche signals

Human embryonic stem cell-derived beta cells offer a promising cell-based therapy for diabetes. However, efficient stem cell to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells, and analyzed their gene expression during development. Our genetic studies reveal the importance of tightly regulated Hedgehog signaling in the pancreatic mesenchyme: inactivation of mesenchymal signaling leads to annular pancreas, whereas stroma-specific activation of signaling via loss of Hedgehog regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through Gli2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and human stem cell cultures significantly promotes insulin-producing cell generation, altogether revealing the requirement for organ-specific regulation of stromal niche signals.

O23 - Effects of systemic glucocorticoid administration on stromal and epithelial GR signaling with consequences for prostate cancer progression.

O23 - Effects of systemic glucocorticoid administration on stromal and epithelial GR signaling with consequences for prostate cancer progression.

Dynamic changes in epithelial cell morphology control thymic organ size during atrophy and regeneration

T lymphocytes must be produced throughout life, yet the thymus, where T lymphocytes are made, exhibits accelerated atrophy with age. Even in advanced atrophy, however, the thymus remains plastic, and can be regenerated by appropriate stimuli. Logically, thymic atrophy is thought to reflect senescent cell death, while regeneration requires proliferation of stem or progenitor cells, although evidence is scarce. Here we use conditional reporters to show that accelerated thymic atrophy reflects contraction of complex cell projections unique to cortical epithelial cells, while regeneration requires their regrowth. Both atrophy and regeneration are independent of changes in epithelial cell number, suggesting that the size of the thymus is regulated primarily by rate-limiting morphological changes in cortical stroma, rather than by their cell death or proliferation. Our data also suggest that cortical epithelial morphology is under the control of medullary stromal signals, revealing a previously unrecognized endocrine-paracrine signaling axis in the thymus.

TGFβ and activin A in the tumor microenvironment in colorectal cancer

Although overall survival in colorectal cancer (CRC) is increasing steadily due to progress in screening, therapeutic options and precise diagnostic tools remain scarce. As the understanding of CRC as a complex and multifactorial condition moves forward, the tumor microenvironment has come into focus as a source of diagnostic markers and potential therapeutic targets.The role of TGFβ in shifting the epithelial cancer compartment towards invasiveness and a pro-migratory phenotype via stromal signaling has been widely investigated. Accordingly, recent studies have proposed that CRC patients could be stratified into distinct subtypes and have identified one poor prognosis subset of CRC that is characterized by high stromal activity and elevated levels of TGFβ. The TGFβ superfamily member activin A is crucial for the pro-metastatic properties of the TGFβ pathway, yet it has been under-researched in CRC carcinogenesis. In this review, we will elucidate the signaling network and interdependency of both ligands in the context of the tumor microenvironment in CRC.

Suppression of Stromal Interferon Signaling by Human Papillomavirus 16.

Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs) and HFKs containing either wild-type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Interferon (IFN)-stimulated genes (ISGs) and extracellular matrix remodeling genes were suppressed, the most prominent pathways affected. STAT1, IFNAR1, IRF3, and IRF7 were knocked down in stromal fibroblasts using lentiviral short hairpin RNA (shRNA) transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated with increased late keratinocyte differentiation but not decreased IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.IMPORTANCE The persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a three-dimensional (3D) model of human epithelium. This finding is significant, because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.

Dissecting the Stromal Signaling and Regulation of Myeloid Cells and Memory Effector T Cells in Pancreatic Cancer.

Myeloid cells are a prominent immunosuppressive component within the stroma of pancreatic ductal adenocarcinoma (PDAC). Previously, targeting myeloid cells has had limited success. Here, we sought to target the myeloid cells through modifying a specific stromal component. A murine model of metastatic PDAC treated with an irradiated whole-cell PDAC vaccine and PDAC specimens from patients treated with the same type of vaccine were used to assess the immune-modulating effect of stromal hyaluronan (HA) degradation by PEGPH20. Targeting stroma by degrading HA with PEGPH20 in combination with vaccine decreases CXCL12/CXCR4/CCR7 immunosuppressive signaling axis expression in cancer-associated fibroblasts, myeloid, and CD8+ T cells, respectively. This corresponds with increased CCR7- effector memory T-cell infiltration, an increase in tumor-specific IFNγ, and improved survival. In the stroma of human PDACs treated with the same vaccine, decreased stromal CXCR4 expression significantly correlated with decreased HA and increased cytotoxic activities, suggesting CXCR4 is an important therapeutic target. This study represents the first to dissect signaling cascades following PDAC stroma remodeling via HA depletion, suggesting this not only overcomes a physical barrier for immune cell trafficking, but alters myeloid function leading to downstream selective increases in effector memory T-cell infiltration and antitumor activity.

Progress on Stromal Cell-related Signaling Pathways in Endometrial Carcinoma Microenvironment

Progress on Stromal Cell-related Signaling Pathways in Endometrial Carcinoma Microenvironment

Effect of Dentin Matrix Components on the Mineralization of Human Mesenchymal Stromal Cells.

This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

Abstract 2032: Investigating the contribution of mTORC1-dependent signaling in the microenvironment to cancer onset in Li-Fraumeni syndrome

Abstract We are investigating the role of stromal mTORC1 signaling in the development of malignancy in individuals with Li-Fraumeni Syndrome (LFS). LFS is a hereditary 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 tumour onset. Chemical inhibitors of the mTORC1 pathway have been shown to promote lifespan extension and delay or even prevent tumour onset in LFS mouse models. These findings are noteworthy, since they suggest that mTORC1 signaling plays a central role in favoring tumorigenesis in LFS. mTORC1 signaling has been implicated in promoting the pro-tumorigenic 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. Mutant p53 has also been shown to promote the secretion of a variety of pro-tumorigenic factors from various cell types. We believe that mTORC1 signaling in LFS stromal cells favors the production and secretion of pro-tumorigenic factors, which may induce changes in the surrounding tissue to promote development of a precancerous niche, thus favoring tumorigenesis. Our objectives are to investigate the contribution of stromal cells and stromal secreted factors in favoring tumorigenesis in LFS, and to investigate the role of mTORC1 signaling in favoring these altered stromal phenotypes. To determine whether patient-derived LFS fibroblasts with TP53 mutations secrete more pro-tumorigenic cytokines than non-LFS fibroblasts, we performed protein arrays with conditioned media (CM) from fibroblast cultures. We saw that LFS fibroblasts secrete markedly higher levels of pro-tumorigenic, SASP-related cytokines. Next, we treated spheroids formed from cancer cells of a common LFS subtype (osteosarcoma) with CM from LFS and non-LFS fibroblast cultures. LFS fibroblast CM had a robust pro-proliferative effect on spheroids, while non-LFS fibroblast CM inhibited spheroid growth. To further address these aims in vivo, we co-injected LFS fibroblasts with cancer cells of a common LFS subtype [rhabdomyosarcoma (RMS)] into Nude mice. We found that tumours formed from the co-injection of RMS cells with LFS fibroblasts grew to be larger than tumours formed from co-injection with non-LFS fibroblasts. Thus far, we have observed that LFS stromal constituents favor cancer cell proliferation in vitro and in vivo. We are currently investigating the role that mTORC1 signaling plays in these processes by repeating these studies following chemical and genetic perturbation of mTORC1 signaling in the LFS fibroblasts. Citation Format: Camilla Giovino, Nish Patel, Sangeetha Paramathas, Gaetano Zafarana, Ran Kafri, David Malkin. Investigating the contribution of mTORC1-dependent signaling in the microenvironment to cancer onset in Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2032.