Stroma-derived Factors Research Articles

Gene Expression Profile of Stromal Factors in Cancer-Associated Fibroblasts from Prostate Cancer

Recent investigations point at the stromal microenvironment to assess additional diagnostic information and provide new therapeutic targets in cancer. The aim of the study was to contribute to the characterization of the phenotype of cancer-associated fibroblasts (CAFs) in prostate cancer (PCa) compared with normal prostate-associated fibroblasts (NAFs) and fibroblasts from benign prostatic hyperplasia (BPH). Three patient populations were prospectively recruited: 23 patients with new localized PCa, 14 patients with advanced PCa treated with androgenic deprivation therapy (ADT), and 7 patients with BPH. Gene expression of 20 stroma-derived factors, including the androgen receptor (AR), chaperones (HSPA1A and HSF1), growth factors (FGF2, FGF7, FGF10, HGF, PDGFB, and TGFβ), proteins implicated in invasion (MMP2, MMP9, and MMP11), inflammation (IL6, IL17RB, NFκB, and STAT3), and in-stroma/epithelium interaction (CDH11, CXCL12, CXCL14, and FAP), was evaluated. Localized PCa CAFs showed a significant higher expression of FGF7, IL6, MMP2, and MMP11 compared with NAFs or IL17RB compared with BPH fibroblasts, but significantly lower expression of FGF10 and IL17RB compared with NAFs or CXCL14 compared with BPH fibroblasts. In addition, CAFs from ADT-resistant PCa showed significantly higher MMP11 and NFκB but significant lower TGFβ expression compared with CAFs from ADT-sensitive tumors. Our results contribute to defining the CAFs phenotypes associated to PCa progression, which may contribute to the diagnosis and design of alternative therapies in PCa.

Human and mouse melanoma cells recapitulate an EMT-like program in response to mesenchymal stromal cells secretome.

The mechanisms underlying the propensity of melanomas to metastasize are not completely understood. We hypothesized that melanoma cells are capable of promptly activating an epithelial-to-mesenchymal transition (EMT)-like profile in response to stroma-derived factors. Thus, we investigated the role of mesenchymal stromal cells (MSCs), a cell population considered as a precursor of tumor stroma, on the activation of an EMT-like profile and acquisition of metastatic traits in melanoma cells. After subcutaneous co-injection with mouse B16 melanoma cells, MSCs occupied perivascular sites within tumors and enhanced B16 metastasis to the lungs. In vitro, MSCs' secretome activated an EMT-like profile in B16cells, reducing their avidity to fibronectin, and increasing their motility and invasiveness. These effects were abrogated upon blocking of MET phosphorylation in B16cells using small molecule inhibitors. MSCs also activated an EMT-like profile in human melanoma cells from different stages of progression. Activation of EMT in human cells was associated with increased levels of p-STAT1 and p-STAT3. In conclusion, both mouse and human melanoma cells are equipped to activate an EMT-like program and acquire metastatic traits through the activation of distinct pathways by MSCs' secretome.

Abstract A62: Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein

Abstract Cancer-associated fibroblasts (CAFs) support tumor growth and metastasis of virtually all solid tumors, making them a potential “universal therapeutic target.” However, there are challenges to designing CAF targeted therapies. First, CAFs are heterogeneous, consisting of tumor-promoting and tumor-restraining populations. Second, molecular targets expressed strictly by tumor-promoting CAFs have yet to be defined. Nonetheless, Fibroblast Activation Protein (FAP) is selectively overexpressed on tumor-promoting CAFs. Moreover, deleting FAP+ cells inhibits tumor growth in preclinical models due to reductions in stroma-dependent signals that support tumor cell survival, growth, and progression and in stroma-derived immunosuppressive factors and can enhance efficacy of other therapies when given in combination. Prior studies in preclinical models suggest, however, that selective elimination of FAPhigh cells while sparing normal FAP+ cells that typically express lower levels of FAP may be necessary to avoid toxicity due to on-target, off-tumor effects. Thus, the aim of this study was to generate a FAP-chimeric antigen receptor (CAR) that selectively targets FAPhigh cells but that exhibits minimal activity against FAPlow cells for use in future clinical trials. We generated a FAP-CAR containing a scFv based on a monoclonal antibody (mAb) we generated that reacts with human and mouse FAP, the CD8 transmembrane region, and the CD3z and 41BB cytoplasmic domains. We show that primary human T cells transduced with this novel FAP-CAR exhibit strong reactivity against cells expressing high levels of human or mouse FAP but not FAPlow or FAP-negative cells. Moreover, these FAP-CART cells inhibited the growth of human FAP-negative A549 tumor xenografts infiltrated by FAP+ murine CAFs in NSG mice. Based on these characteristics, this novel FAP-CAR will be developed for use in clinical trials for human cancer patients. Citation Format: Estela Noguera-Ortega, Leslie Todd, James Monslow, Jing Sun, Steven M. Albelda, Ellen Pure. Inhibition of tumor growth by novel CART redirected against cells expressing high levels of fibroblast activation protein [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A62.

Abstract 1233: Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity

Abstract In cancer, suppression of host immunity is related, in part, to immature myeloid-derived suppressor cells (MDSCs) expanded in response to various tumor- or stroma-derived factors. Here, the expression and activity of myeloperoxidase (MPO) was examined in both granulocytic (PMN) and monocytic (M) MDSC subsets recovered from the spleens of 4T1 tumor-bearing mice, as well as interferon regulatory factor-8 (IRF8) knockout mice (IRF8-/-) which develop high levels of MDSCs due to IRF8-deficiency. Phenotypically identical cells from non-tumor-bearing mice served as controls. In addition, the role of MPO in PMN-MDSCs as a determinant of DNA damage induced by the anticancer agents etoposide (VP-16), camptothecin (CPT) and parthenolide (PTL), was evaluated for potential immunomodulatory effects. MPO protein content was ~4-fold greater in PMN-MDSCs from tumor-bearing compared to non-tumor-bearing mice, while MPO specific activity was equivalent in these two populations. MPO protein content in PMN-MDSCs from IRF8-/- mice was at least comparable to levels found in tumor-bearing hosts. In both IRF8-/- and tumor-bearing mice, PMN-MDSCs contained 5-10-fold greater MPO protein and overall enzyme activity compared to isolated M-MDSCs. VP-16 (5 µM)- and PTL (100 µM)-induced DNA strand breaks were 4-fold greater in PMN- compared to M-MDSCs from tumor-bearing mice. In addition, the heme-synthesis inhibitor, succinylacetone (SA) reduced MPO activity 2-3-fold in PMN-MDSCs from tumor-bearing mice with a corresponding decrease in drug-induced DNA damage for both VP-16 and PTL with no effect on CPT-induced DNA damage. The antioxidant dehydroascorbate decreased VP-16-induced DNA damage in PMN-MDSCs containing MPO activity, but not in those cells pre-incubated with SA to deplete MPO. These results indicate that MPO-dependent activation of VP-16 and PTL enhance DNA damage. Antioxidant protection against VP-16-induced DNA damage is consistent with MPO-mediated oxidative activation of VP-16 to a redox-cycling phenoxyl radical (Mol. Pharmacol. 79: 479-487, 2011). Overall, these results suggest that: 1) PTL may be useful to eliminate PMN-MDSCs; 2) MPO activation of VP-16 in PMN-MDSCs may be deleterious via oxidative DNA damage resulting in mixed-lineage leukemia (MLL) gene rearrangements known to be causal for therapy-related myeloid leukemias. Support: CA172105; CA090787; CA140622 Citation Format: Jack C. Yalowich, Ragu Kanagasabai, Colleen S. Netherby, Michelle N. Messmer, Scott I. Abrams. Myeloperoxidase (MPO) activity in myeloid-derived suppressor cells (MDSCs): effects on anticancer drug activity [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 1233.

Abstract LB-214: The immunosuppressive roles of ovarian stromal tumor microenvironment

Abstract Ovarian cancer is the most lethal gynecologic malignancy in the US. Our group and others has shown that CD8+ lymphocyte infiltration in the ovarian tumor epithelium is associated with prolonged survival in patients with high-grade serous ovarian cancer. Despite the increasing evidence on stromal involvement in tumor progression, the interactions between stromal fibroblasts and cancer cells, as well as the underlying genetic composition of the stromal cells that could regulate the infiltration and activation of CD8+ cytotoxic T lymphocytes (CTLs) and affect patient survival is not fully understood. The present study seeks to evaluate the roles and to delineate the underlying mechanisms by which stromal cancer associated fibroblasts (CAFs) modulates immune response in ovarian cancer, particularly immune suppression by CAF-derived protein factors. By laser microdissection and transcriptome profiling of tumor tissue samples from ovarian cancer patients, we identified CAF-specific gene signatures for ovarian cancer that are associated with the survival duration of patients and with the differential tumor immune response. Among the differentially expressed genes identified, validation study by qPCR and immunohistochemistry indicated that stromal MFAP5 expression is significantly higher in patients with short survival duration and in patients with lower intratumoral CD8+ T cell density. Survival analysis result suggested that high stromal MFAP5 expression indicates poor disease prognosis (Hazard ratio=2.722, P<0.001, N=102) and multiplex immunofluorescent staining result suggested that high stromal MFAP5 expression is associated with lower density of CD8+ / Granzyme B+ activated tumor-infiltrating CTLs (P=0.006). Our in vitro data suggested that stromal MFAP5 promotes ovarian cancer cell invasion, migration and chemoresistance. At the same, we showed that MFAP5 modulates the expression of immune-related genes and generate an immunosuppressive microenvironment through suppression of CD8+ T cell activation in the ovarian tumor tissue. Cell culture experiment results showed that MFAP5 protein treatment induced upregulation of CD47, an immune suppressive protein, in cancer cells and CD8+ T cells. Using an ovarian cancer-bearing immune competent animal model, we demonstrated that inhibition of stromal MFAP5-mediated signaling cascade suppressed tumor progression and promoted tumor immune response. Delineating the molecular mechanism by which MFAP5 modulates the progression and immune responses of ovarian cancer will shed light on the design of novel treatment modalities based on the blockade of tumor stroma-derived factors, which could promote activation and trafficking of cytotoxic CD8+ T cells and improve patient survival rates. Citation Format: Tsz-Lun Yeung, Cecilia S. Leung, Kwong-Kwok Wong, Michael J. Birrer, Stephen T. Wong, Karen H. Lu, Samuel C. Mok. The immunosuppressive roles of ovarian stromal tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2017-LB-214

Stromal factors involved in human prostate cancer development, progression and castration resistance.

To detect new predictive markers from the prostate cancer tissue, to study the expression by cultured cancer-associated fibroblasts (CAFs) of stromal factors implicated in prostate carcinogenesis, and to compare their expressions in localized, metastatic, castration-sensitive (CSCP), castration-resistant prostate tumors (CRCP) as well as in fibroblasts from benign prostatic hyperplasia (BPH). The genomic expression of 20 stroma-derived factors, including the androgen receptor (AR), growth factors (FGF2, FGF7, FGF10, HGF, TGFβ, PDGFB), protein implicated in invasion (MMP-2, MMP-9 and MMP-11), inflammation (IL-6, IL-17, STAT-3 and NFκB), stroma/epithelium interaction (CDH11, FAP, CXCL12 and CXCL14) and chaperones (HPA1A and HSF1), was evaluated in cultured fibroblasts both from BHP and prostate carcinomas (PCa). After isolation and culture of fibroblasts by biopsy specimens, RNA was isolated and genomic studies performed. Finally, 5 BPH and 37 PCa specimens were selected: clinically localized (19), metastatic (5), CSCP (7) and CRPC (6). Interleukin-17 receptor (IL-17RB) was highly expressed in CAFs compared with fibroblasts from BPH. However, metalloproteinase-2 and chemokine ligand 14 (CXCL14) were expressed at higher levels by fibroblasts from BPH. The fibroblastic growth factor-7 was highly expressed by CAFs from localized tumors, but metalloproteinase-11 in metastatic tumors. MMP-11, androgen receptor (AR) and heat-shock-70kda-protein-1A (HSPA1A) expressions were significantly higher in CAFs from CRPC. These results demonstrate a CAFs heterogeneity among prostate carcinomas with regard to some molecular profile expressions that may be relevant in tumor development (IL-17RB), progression (MMP-11) and castration resistance (AR, MMP-11 and HSPA1A).

Abstract IA04: Stromal regulation of pancreatic cancer epigenome and metabolome

Abstract A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the dramatic accumulation of stromal components implicated in tumor growth, metastasis, and resistance to therapy. This creates a living chimera in which stroma-derived growth factors, matrix proteins and inflammatory cytokines are thought to nurture the nascent tumor. This study reveals that the stromal secretome exerts rapid epigenomic changes in the cancer cells that effectively marshals up metabolic and genomic networks needed to power cell growth. To tackle the complexity of the tumor microenvironment (TME), we devised a three-dimensional culture system to dissect the adaptive impact of the stromal secretome on the cancer cell. This integrative approach identified the rapid mobilization of more than a dozen gene networks (cell cycle, steroid biosynthesis, metabolic pathways, DNA replication, etc.), as well as a commensurate adaptive metabolome. A major unanswered question relates to the sequence of events that result in the formation and ultimate function of these networks, in part generated by rapid changes in acetylation of histone H3 at lysines 9 and 27 (H3K9ac and H3K27ac). Our work suggests the existence of a molecular and-gate such that tumor activation is the consequence of mutant Ras and the tumor micro-environment. To mediate this molecular hand-shake, we find that BRD2 acts in the epithelial compartment to transduce stromal cues at the genomic level. Based on the above, and consistent with recent Mazur PK et al Nat Med (2015), we show the BET bromodomain inhibitor JQ1 targets a key synergy point and reduces pancreatic tumor growth in vivo. Collectively, this work describes a novel connection between the stromal response and a driver oncogene and may offer new insights into the role of the TME in the treatment of ras-driven tumor. Citation Format: Mara H. Sherman, Ronald Evans. Stromal regulation of pancreatic cancer epigenome and metabolome. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA04.

Tumor-derived factors modulating dendritic cell function.

Dendritic cells (DC) play unique and diverse roles in the tumor occurrence, development, progression and response to therapy. First of all, DC can actively uptake tumor-associated antigens, process them and present antigenic peptides to T cells inducing and maintaining tumor-specific T cell responses. DC interaction with different immune effector cells may also support innate antitumor immunity, as well as humoral responses also known to inhibit tumor development in certain cases. On the other hand, DC are recruited to the tumor site by specific tumor-derived and stroma-derived factors, which may also impair DC maturation, differentiation and function, thus resulting in the deficient formation of antitumor immune response or development of DC-mediated tolerance and immune suppression. Identification of DC-stimulating and DC-suppressing/polarizing factors in the tumor environment and the mechanism of DC modulation are important for designing effective DC-based vaccines and for recoveryof immunodeficient resident DC responsible for maintenance of clinically relevant antitumor immunity in patients with cancer. DC-targeting tumor-derived factors and their effects on resident and administered DC in the tumor milieu are described and discussed in this review.

Targeting Stromal-Cancer Cell Crosstalk Networks in Ovarian Cancer Treatment.

Ovarian cancer is a histologically, clinically, and molecularly diverse disease with a five-year survival rate of less than 30%. It has been estimated that approximately 21,980 new cases of epithelial ovarian cancer will be diagnosed and 14,270 deaths will occur in the United States in 2015, making it the most lethal gynecologic malignancy. Ovarian tumor tissue is composed of cancer cells and a collection of different stromal cells. There is increasing evidence that demonstrates that stromal involvement is important in ovarian cancer pathogenesis. Therefore, stroma-specific signaling pathways, stroma-derived factors, and genetic changes in the tumor stroma present unique opportunities for improving the diagnosis and treatment of ovarian cancer. Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor stroma that have demonstrated supportive roles in tumor progression. In this review, we highlight various types of signaling crosstalk between ovarian cancer cells and stromal cells, particularly with CAFs. In addition to evaluating the importance of signaling crosstalk in ovarian cancer progression, we discuss approaches that can be used to target tumor-promoting signaling crosstalk and how these approaches can be translated into potential ovarian cancer treatment.

Stroma-Derived Factors Significantly Impact the Drug Response Profiles of Patient-Derived Primary AML Cells: Implications for Drug Sensitivity Testing

Background:Bone marrow (BM) microenvironment plays an important role in development of drug resistance in acute myeloid leukemia (AML) by supporting survival of therapy resistant leukemic cells that eventually may lead to disease relapse. Consideration of tumor stroma factors is therefore critically important when assessing the efficacy of drugs in the ex vivo drug testing of primary AML cells. To study the effects of tumor stroma factors on the response of AML patient cells ex vivo to a panel of cancer drugs, we evaluated the effects of stroma-derived conditioned medium (CM) on the sensitivity of genomically defined primary AML cells.Methods:Primary AML cells were isolated by Ficoll gradient separation from BM aspirates or peripheral blood of AML patients (n=13). For drug testing, 303 small molecule inhibitors were plated on 384-well plates in 5 different concentrations over a 10,000-fold concentration range. The stromal-cell conditioned medium (CM) was made by culturing human bone marrow (BM) stromal cell line HS-5 (American Type Culture Collection) in RPMI 1640 medium for 3 days. AML cells were added to the plates in either CM diluted with RPMI 1640 medium (25% CM) or in mononuclear cell medium (MCM, Promocell), which was used as the standard medium comparison. Cell viability was measured after 72 h and dose response curves generated for each drug. Drug sensitivity scores (DSS) were calculated as described previously (Yadav et al, 2014). Phosphorylation profiles of 43 proteins were analyzed with a human phospho-kinase array (R&D Systems). In addition, somatic mutations were identified by exome sequencing using DNA from the leukemia cells and matched skin biopsies, while expressed fusion genes were identified by transcriptome sequencing.Results:AML samples with activating mutations to kinases such as FLT3 or PDGFRB exhibited more sensitive ex vivo drug response profiles, particularly to broad-spectrum kinase inhibitors, compared to samples driven by other types of mutations. When the same AML samples were compared between the two conditions, CM or MCM, the drug sensitivities were different for many classes of drugs (Table 1). In CM, samples typically lost sensitivity to many of the tested drugs, such as topoisomerase II inhibitors, BCL2 inhibitors and several other classes of tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity in CM was particularly striking in the FLT3 and PDGFRB mutated cases. Cluster analysis of overall drug responses for AML samples tested in MCM resulted in a tight group of most TKIs, reflecting their overlapping target profiles. However, when the analysis was applied to responses from the same cells tested in CM, the TKI grouping was more dispersed. Thus, these results indicate that tyrosine kinase signaling is stringently regulated in standard medium, whereas CM helps to support cell survival resulting in lower responses to a range of TKIs. To test this hypothesis, phosphorylation of 43 different kinases was measured with AML samples incubated in either CM or MCM. CM induced phosphorylation of multiple proteins including p38α, HSP27, Src, Lyn, Hck and STAT6 proving the activation of other signaling pathways.Conclusions:Our dataindicate that stromal cell conditioned medium may have prominent effects on ex vivo drug responses of AML cells. BM factors likely provide survival cues that make primary patient-derived AML cells resistant to several targeted agents, such as topoisomerases and TKIs. This underscores the need to develop drug testing methods that take into account tumor-microenvironment interactions. [Display omitted] DisclosuresGjertsen:BerGenBio AS: Membership on an entity’s Board of Directors or advisory committees; Boehringer Ingelheim : Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Kallioniemi:Medisapiens: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Wennerberg:Pfizer: Research Funding. Heckman:Celgene: Research Funding.

Tumor stroma-derived factors skew monocyte to dendritic cell differentiation toward a suppressive CD14+ PD-L1+ phenotype in prostate cancer

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C‒C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor β (TGFβ) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+ T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findings in vitro, tumor-infiltrating CD14+ cells in situ were found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+ T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa.

Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel insights into the molecular signatures and the modulatory role of colon cancer associated fibroblasts, and establish a valuable resource for the development of therapeutic agents or novel clinic biomarker.

P1-03-03: Adaptive Exploitation of Stromal Cell Metabolism by Tumor Cells.

Abstract Tumor cells secrete cytokines to recruit and activate stromal cells in the tumor milieu leading to reciprocal paracrine support of tumor growth by stroma-derived growth factors. This is an important means by which tumors adapt their microenvironment to facilitate their growth. Indeed, breast cancer development and metastatic progression is highly dependent on stromal support, particularly from carcinoma associated fibroblasts (CAFs). As a result of aerobic glycolysis, tumor cells produce and secrete high levels of lactate, thought to be a toxic byproduct that needs to be extruded into the tumor milieu. Using CAFs generated by prolonged exposure to tumor conditioned medium, we have investigated the role that lactate may play in CAF-mediated support of tumor growth. In addition to extruding lactate as a byproduct of glycolysis, we suggest that tumor cells secrete it to recruit and subsequently exploit stromal cells to recycle lactate into utilizable metabolites, such as pyruvate, to fulfill metabolic demands of tumor cells. Our hypothesis is that lactate secreted by tumor cells is taken up by stromal cells and converted to metabolites critical for tumor growth and progression. Our studies indicate that (i) MDA-MB-231 breast cancer cells secrete significantly higher levels of lactate (3-fold more) under hypoxia, and that (ii) lactate recruits mesenchymal stem cells, the precursors of CAFs, towards tumor cells by activating signaling pathways to enhance migration. Lactate is transported by a family of proteins termed monocarboxylate transporters (MCTs); cells take up lactate via MCT1 and efflux it via MCT4. Expectedly, MDA-MB-231 breast carcinoma cells display low expression of MCT1 while exhibiting high expression of MCT4. However, CAFs show high expression of MCT1 while displaying low expression of MCT4, indicating that lactate extruded by the tumor cells is taken up by stromal cells. Our investigation further revealed that expression of lactate dehydrogenase B and pyruvate dehydrogenase are induced upon lactate exposure in CAFs, supporting the contention that the lactate taken up by stromal cells is metabolized. This implies that CAFs may be less glycolytic than MDA-MB-231s. Indeed the expression of pyruvate kinase isoform 2 (PKM2), which positively correlates with glycolysis, is greater in MDA-MMB-231s than in CAFs, while CAFs express significantly more PKM1, the isoform that correlates with a more oxidative metabolic state. Finally, 13C NMR spectroscopic analyses indicate that 13C-lactate is indeed metabolized via the TCA cycle in stromal cells, further supporting our hypothesis. Thus, stromal fibroblasts in the tumor microenvironment have the capacity to take up tumor-secreted lactate and use it as an energy source. To our knowledge this is the first in vitro model system demonstrating tumor/stroma metabolic coupling by which tumor cells exploit stromal cells. A better understanding of the molecular mechanisms governing metabolic cooperation within the tumor milieu will potentially identify new targets for therapeutic intervention. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-03.

Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Abstract LB-287: Adaptive exploitation of stromal cell metabolism by tumor cells

Abstract Tumor cells secrete cytokines to recruit and activate stromal cells in the tumor milieu leading to reciprocal paracrine support of tumor growth by stroma-derived growth factors. This is an important means by which tumors adapt their microenvironment to facilitate their growth. Indeed, breast cancer development and metastatic progression is highly dependent on stromal support, particularly from carcinoma associated fibroblasts (CAFs). As a result of aerobic glycolysis, tumor cells produce and secrete high levels of lactate, thought to be a toxic byproduct that needs to be extruded into the tumor milieu. Using CAFs generated by prolonged exposure to tumor conditioned medium, we have investigated the role that lactate may play in CAF-mediated support of tumor growth. In addition to extruding lactate as a byproduct of glycolysis, we suggest that tumor cells secrete it to recruit and subsequently exploit stromal cells to recycle lactate into utilizable metabolites, such as pyruvate, to fulfill metabolic demands of tumor cells. Our hypothesis is that lactate secreted by tumor cells is taken up by stromal cells and converted to metabolites critical for tumor growth and progression. Our studies indicate that (i) MDA-MB-231 breast cancer cells secrete significantly higher levels of lactate (3-fold more) under hypoxia, and that (ii) lactate recruits mesenchymal stem cells, the precursors of CAFs, towards tumor cells by activating signaling pathways to enhance migration. Lactate is transported by a family of proteins termed monocarboxylate transporters (MCTs); cells take up lactate via MCT1 and efflux it via MCT4. Expectedly, MDA-MB-231 breast carcinoma cells display low expression of MCT1 while exhibiting high expression of MCT4. However, CAFs show high expression of MCT1 while displaying low expression of MCT4, indicating that lactate extruded by the tumor cells is taken up by stromal cells. Our investigation further revealed that expression of lactate dehydrogenase B and pyruvate dehydrogenase are induced upon lactate exposure in CAFs, supporting the contention that the lactate taken up by stromal cells is metabolized. Finally, 13C NMR spectroscopic analyses indicate that 13C-lactate is indeed metabolized via the TCA cycle in stromal cells, further supporting our hypothesis. Thus, stromal fibroblasts in the tumor microenvironment have the capacity to take up tumor-secreted lactate and use it as an energy source. To our knowledge this is the first in vitro model system demonstrating tumor/stroma metabolic coupling by which tumor cells exploit stromal cells. A better understanding of the molecular mechanisms governing metabolic cooperation within the tumor milieu will potentially identify new targets for therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-287. doi:10.1158/1538-7445.AM2011-LB-287

A Macromolecular Signaling Complex Formed by CXCR4, VLA4 and hERG1 K+ Channels Mediates Bone Marrow-Induced Chemo-Resistance in Childhood Acute Lymphoblastic Leukemias: Shortcoming Effects of hERG1 Channels Inhibitors.

Childhood Acute lymphoblastic leukemia (ALL) has experienced a dramatic improvement in survival rates over the past 40 years. At this time, the likelihood of cure is greater than 70%. Despite such success, relapse still occurs in 25–30% of children. The major cause of treatment failure is intrinsic or acquired drug resistance. It has been reported that stromal cells of the bone marrow (BMSC) provide a sanctuary in which subpopulations of leukaemia cells can evade chemotherapy-induced death and acquire a drug-resistant phenotype. This explains why, when ALL cell lines are cultured on human BMSC, the apoptotic effect of chemotherapic drugs is strongly inhibited. The mechanisms of BMSC-mediated protection involve a complex interplay among stroma-derived factors, in particular the SDF-1a and the chemokine receptor CXCR4. For these reasons, CXCR4-targeting compounds are nowadays considered potential therapeutic tools in ALL (Burger JA and Burkle A, Br J Haematol 137: 288–296, 2007). An additive mechanism of BMSC-induced protection involeves the interaction between adhesion receptors on leukaemia cells and adhesion molecules, like fibronectin, on the surface of BMSC (Tabe Y et al, Cancer Res 67:1238–1250, 2007). Recent evidence indicates that integrins trigger survival signals since they form macromolecular complexes with plasma membrane proteins. A novel integrin partner in such complexes is represented by ion channel proteins. The channel protein is not merely a bystander interactor, but it often feeds back by controlling integrin activation and downstream signaling (Arcangeli A et al, Trends Cell Biol 16:631–639, 2006). This mechanisms can give a molecular explanation to the recent evidences that ion channels, especially K+ channels, mark and regulate specific stages of cancer progression, and hence may represent novel targets for cancer therapy. (Becchetti A. and Arcangeli A., J. Gen. Physiol., 28: 313–314, 2008). Among ion channels, hERG1 channels, are capable of forming multiprotein complexes with integrins in tumor cells. We report here the discovery of a peculiar multiprotein complex formed by CXCR4 and the b1 integrin subunit, besides hERG1 channels, occurring in B-ALL cells. The assembly of the complex is stimulated by adhesion onto BMSC, and is further modulated by SDF-1a. Furthermore, the hERG1/beta1/CXCR4 complex triggers the activation of different intracellular signalling pathways (MAPK activation, pAKT phosphorylation). All the BMSC-induced signalling pathways are strongly inhibited by hERG1 channel blockers. Finally, when cultured on BMSC, all the B-ALL cell lines experienced a strong reduction of apoptosis after treatment with chemotherapic drugs (doxorubicin, prednisone, L-asparaginase, metothrexate). The addition of specific hERG1 blockers bypasses drug resistance, promptly restoring a significant apoptosis in leukemia cells. On the whole, our results allow to include hERG1 channels in a scheme where they act as upstream regulators of integrin/chemokine receptors pro survival signals. In addition data here presented indicate a potential role for hERG1 inhibitors as a novel strategy for overcoming drug resistance in the treatment of ALL.

Stromal–epithelial interactions in skin homeostasis, wound repair and skin cancer

Stromal–epithelial interactions in skin homeostasis, wound repair and skin cancer

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.

Macrophage HIV-1 infection and the gastrointestinal tract reservoir.

Excluding parenteral transmissions, virtually all vertical and homosexual transmission of human immunodeficiency virus type 1 (HIV-1) occurs via the gastrointestinal tract. Cellular routes implicated in the translocation of virus across the epithelium include M cells, dendritic cells, and epithelial cells. Intestinal epithelial cells express CCR5 and can selectively transfer CCR5-tropic HIV-1, the phenotype of the majority of transmitted viruses. In the lamina propria, virus encounters the largest reservoir of mononuclear cells in the body. Surprisingly, lamina propria lymphocytes, not macrophages, express CCR5 and CXCR4 and support HIV-1 replication, implicating intestinal lymphocytes as the initial target cell in the intestinal mucosa. From the mucosa, virus is disseminated to systemic sites, followed by profound depletion of CD4+ T cells, first in the intestinal lamina propria and subsequently in the blood. As mucosal and circulating CD4+ T cells are depleted, monocytes and macrophages assume an increasingly important role as target and reservoir cells for HIV-1. Blood monocytes, including HIV-1-infected cells, are recruited to the mucosa, where they differentiate into lamina propria macrophages in the presence of stroma-derived factors. Although the prevalence of HIV-1-infected macrophages in the mucosa is low (0.06% of lamina propria mononuclear cells), the extraordinary size of the gastrointestinal mucosa imparts to intestinal macrophages a prominent role as a HIV-1 reservoir. Elucidating the immunobiology of mucosal HIV-1 infection is critical for understanding disease pathogenesis and ultimately for devising an effective mucosal HIV-1 vaccine.

Active form of Notch members can enforce T lymphopoiesis on lymphoid progenitors in the monolayer culture specific for B cell development.

The in vitro induction of T lymphopoiesis needs the precise stereoscopic structure of thymus tissues as seen in fetal thymus organ culture. In this study, we demonstrated for the first time that the introduction of the intracellular region of Notch1 can induce T cells expressing TCR without any thymic environment. In the coculture on the monolayer of OP-9, which was originally known to support B cell specific development, hemopoietic progenitors developed into Thy-1(+)CD25(+) T lineage cells if the progenitor cells were infected with the retrovirus containing Notch1 intracellular domains. The Thy-1(+) cells progressed to a further developmental stage, CD4 and CD8 double-positive cells expressing TCR on the cell surface, if they were further cultured on OP-9 or in the thymus. However, T cell induction by intracellular Notch1 failed unless both OP-9 and IL-7 were present. It is notable that Notch2 and Notch3 showed an effect on T lymphopoiesis similar to that of Notch1. These results indicate that in vitro T lymphopoiesis is inducible by signaling via Notch family members in a lineage-specific manner but shares other stroma-derived factors including IL-7 with B lymphopoiesis.