Changes In Tumor Microenvironment Research Articles

Therapy-induced microenvironmental changes in cancer.

In the past decade, the focus of tumor biology research has been switching from the functional dissection of oncogenes and tumor suppressor genes to investigation of the cross-talk between tumor cells and their microenvironment. Tumorigenesis requires the organized assembly of cancer cells with non-malignant cells and non-cellular stroma, resembling an abnormal organogenesis. This process can be modulated by local cellular stress responses, such as senescence, ER stress and autophagy, and inflammatory and immunosuppressive cells and effector molecules within the tumor microenvironment (TME). Various cellular stress responses and cell death modalities are triggered in response to chemotherapies, radiotherapies, and targeted therapies (including immunotherapies). The exposure of immunostimulatory factors could (re)awaken anti-tumor immunity. Unexpectedly, the gut microbial flora is becoming recognized as an important external modulator of the TME. We will discuss in detail the TME changes that take place after certain cancer therapies, highlighting the importance of cellular stress responses, tumor-infiltrating immune cells, and microbiota-derived factors.

Abstract PL07-02: Tumor hypoxia and radiation resistance: Anything new on the horizon?

Abstract Hypoxia is one of the key factors influencing clinical outcome after radiotherapy responsible for reduced local control that will influence overall survival, as may the hypoxic conditions by increasing malignant progression. In sharp contrast with the progresses and the rapid diffusion of functional imaging to characterize hypoxic tumors and the fact that experimental correction of tumor tumor hypoxia reverses tumor resistance to irradiation, there is no approved concomitant therapy in order to maximize response to radiotherapy for the treatment of hypoxic tumors. For decades, hypoxia was thought to act primarily on intrinsic tumor cells resistance, namely the number of clonogenic cancer stem cells surviving after radiation treatment. In the meantime, improvements into the understanding of the mechanisms triggered by hypoxia have unveiled novel molecular targets for potential combination to radiation therapy such as the interplay between STAT3 signaling and adaptation to hypoxia while the impact of tumor hypoxia on tumor microenvironment changes have expand to the immune network. Increased cellular turnover and hypoxia promote the production and release of large amounts of immunosupressive adenosine into the local microenvironment. Hypoxia can induce HIF-1a-dependent expression of arginase-1 and M2 polarization of macrophages. The spectrum of candidate therapies to target hypoxic tumors has moved from bioreductive cytotoxic agents to HIF-1, STAT3 inhibitors while growing number of immunological tools are becoming available for targeting the immune component. The ongoing EORTC 1219-ROG-HNCG/DAHANCA project 29 has gained form the past clinical attempts to target hypoxic tumors and will provide the methodology way for future clinical development of such novel combos based on optimal patients selection using biological and/or imaging criteria. Citation Format: Eric Deutsch, CYrus Chargari. Tumor hypoxia and radiation resistance: Anything new on the horizon? [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-02.

Diminution of miR-340-5p levels is responsible for increased expression of ABCB5 in melanoma cells under oxygen-deprived conditions

Melanoma is usually highly refractory to chemotherapy. This resistance to treatment is mainly due to high heterogeneity and plasticity of melanoma cells strictly connected to changes in tumor microenvironment. Hypoxia can drastically alter cancer biology. Solid tumor cells under hypoxia gain stem-like features, they are more invasive and drug-resistant than their normoxic counterparts. These effects could be mediated by changes in miRNA expression under hypoxia. MiRNAs are small non-coding RNA molecules that can negatively control gene expression. In the present study using microarray technology we evaluated the expression of miRNAs in melanoma cells derived from nodular melanoma and grown under normoxic and hypoxic conditions. Using R environment for statistical analysis we found that 70 miRNAs were differentially-expressed, and 16 of them were significantly down-regulated in melanoma cells grown in hypoxic conditions compared to cells grown in normoxia. We intended to find transcripts whose expression is increased due to down-regulation of selected miRNAs. Bioinformatics analysis revealed that increased levels of HIF-2α, ABCB5, OCT4, SOX2 and ZEB1 in different melanoma populations under hypoxia could be a result of significant down-regulation of miR-340-5p. Inhibition of miR-340-5p confirmed that this miRNA negatively influences the expression of ABCB5. This is the first study showing the relationship between miR-340-5p and expression of ABCB5, a transmembrane transporter involved in drug resistance considered as a marker of melanoma stem-like cells.

Tumor Microenvironment in Head and Neck Squamous Cell Carcinomas.

Recent studies about solid tumors demonstrated that tumor microenvironment has an important role in tumor progression, aggressivity, and metastasis process, in addition to genetic aberrations and molecular alterations of cancer cells. Therefore, the crosstalks between cancerous and noncancerous cells and metabolic changes in tumor microenvironment cause significant detrimental effects. The purpose of this review is to present the role and effect of noncancerous cells and their crosstalks with cancer cells, metabolic changes in tumor microenvironment, and to discuss the clinical significance of all these factors with respect to the current literature.

Abstract A04: Aging microenvironment modulates melanoma invasion and metastasis

Abstract The incidence of melanoma rises dramatically after the age of 55. Due to an increase in aging population, it is important to study the change in molecular mechanisms due to aging that would allow development of therapies that are tailored to the age of the patients. The role of the tumor microenvironment in modulating cancer characteristics is widely recognized and it also provides targets for therapeutic intervention. Due to this, we hypothesized that changes in tumor microenvironment due to aging could affect the progression of the melanoma. We obtained skin fibroblasts from healthy donors aged 25-35, as well as skin fibroblasts from healthy donors aged 55-65. We cultured these fibroblasts and used conditioned media from them to affect the invasion of melanoma cells in 3D spheroid invasion assays, where aged fibroblasts promoted invasion of melanoma cells into collagen. We also built artificial skin (reconstructs) using young and aged fibroblasts and demonstrated that skin built with aged fibroblasts promoted melanoma cell invasion. Finally using a transgenic mouse model of melanoma (Yumm1.7, BRAFV600E/PTEN-/-) we observed that the injection of melanoma cells into the tail vein of aged mice (52 weeks) formed metastastic colonies much more rapidly than those injected into the tail vein of young mice (8 weeks). To study the factors involved in the aging microenvironment, we performed a proteomics study of the secretome from young and aged fibroblasts. From this study, we observed that aged fibroblasts secreted inhibitors of canonical Wnt signaling, as well as increased deposition of extracellular matrix components in the aging microenvironment. Since inhibition of canonical Wnt signaling has been linked to decreased sensitivity towards BRAF inhibitors in melanoma, we injected Yumm 1.7 cells subcutaneously into aged and young mice. These mice were then treated with BRAF inhibitors. We observed an increased resistance in response to therapy in the aged mice. We also prepared skin reconstruct from fibroblasts with knockdown of the proteins that we identified from secretome and treated them with PLX4720. These results indicated the role of these extracellular matrix proteins in melanoma. We are exploring the mechanisms of how these extracellular matrix proteins affect the sensitivity towards chemotherapeutics. We concluded that aging could alter tumor microenvironment thus resulting in increased metastasis and therapy resistance. It is important that the studies in cancer therapies take into account the age of the patient to achieve better response in patients. Citation Format: Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Michael O'Connell, Marie Webster, Jessica Appleton, Phil Cheng, Alexander Valiga, Rachel Morissette, Nazli McDonnell, Luigi Ferrucci, Andrew Kossenkov, Katrina Meeth, Marcus Bosenberg, Hsin-Yao Tang, Xiangfan Yin, William Wood, III, Elin Lehrmann, Kevin Becker, Keith Flaherty, Dennie Frederick, Jennifer Wargo, Katherine Aird, Rugang Zhang, Xiaowei Xu, Qin Liu, David Speicher, Ashani Weeraratna. Aging microenvironment modulates melanoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A04.

Fusion with human lung cancer cells elongates the life span of human umbilical endothelial cells and enhances the anti-tumor immunity.

Human umbilical endothelial cells (HUVECs) have been proved as an effective whole-cell vaccine inhibiting tumor angiogenesis. However, HUVECs divide a very limited number of passages before entering replicative senescence, which limits its application for clinical situation. Here, we fused HUVECs with human pulmonary adenocarcinoma cell line A549s and investigated the anti-tumor immunity of the hybrids against mice Lewis lung cancer. HUVECs were fused with A549s using polyethylene glycol and were sorted by flow cytometry. The fusion cells (HUVEC-A549s) were confirmed by testing the expression of telomerase and VE-cadherin, the senescence-associated β-galactosidase activity, and tube formation ability. HUVEC-A549s were then irradiated and injected into the C57BL/6 mice of protective, therapeutic, and metastatic models. The mechanism of the anti-tumor immunity was explored by analyzing mice sera, spleen T lymphocytes, tumor microenvironment, and histological changes. HUVEC-A549s coexpressed tumor and endothelial markers and maintained the vascular function of tube forming at passage 30 without showing signs of senescence. HUVEC-A549s could induce protective and therapeutic anti-tumor activity for LL(2) model and presented stronger activity against metastasis than HUVECs. Both humoral and cellular immunity were participated in the anti-angiogenic activity, as HUVECs-neutralizing IgG and HUVECs-toxic lymphocytes were increased. Angiogenic mediators (VEGF and TGF-β) and tumor microenvironment cells MDSCs and Tregs were also diminished. Our findings might provide a novel strategy for HUVECs-related immunotherapy, and this vaccine requires lower culture condition than primary HUVECs while enhancing the anti-tumor immunity.

Quantitative changes in tumor-associated M2 macrophages characterize cholangiocarcinoma and their association with metastasis.

The tumor microenvironment (TME) includes numerous non-neoplastic cells such as leukocytes and fibroblasts that surround the neoplasm and influence its growth. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are documented as key players in facilitating cancer appearance and progression. Alteration of the macrophage (CD68, CD163) and fibroblast (α-SMA, FSP-1) cells in Opisthorchis viverrini (Ov)-induced cholangiocarcinoma (CCA) was here assessed using liver tissues from an established hamster model and from 43 human cases using immunohistochemistry. We further investigated whether M2-activated TAMs influence CCA cell migration ability by wound healing assay and Western blot analysis. Macrophages and fibroblasts change their phenotypes to M2-TAMs (CD68+, CD163+) and CAFs (α-SMA+, FSP-1+), respectively in the early stages of carcinogenesis. Interestingly, a high density of the M2-TAMs CCA in patients is significantly associated with the presence of extrahepatic metastases (p=0.021). Similarly, CD163+ CCA cells are correlated with metastases (p=0.002), and they may be representative of an epithelial-to-mesenchymal transition (EMT) with increased metastatic activity. We further showed that M2-TAM conditioned medium can induce CCA cell migration as well as increase N-cadherin expression (mesenchymal marker). The present work revealed that significant TME changes occur at an early stage of Ov-induced carcinogenesis and that M2-TAMs are key factors contributing to CCA metastasis, possibly via EMT processes.

Mechanism of Warburg effect and its effect on tumor metastasis

肿瘤细胞葡萄糖代谢的一个特点就是在氧含量正常的情况下依然选择利用糖酵解，即Warburg效应。Warburg效应的内在机制十分复杂，可能与癌基因激活、抑癌基因失活，糖代谢酶表达异常和肿瘤微环境改变等有关，具体的机制还有待进一步研究。Warburg效应这种代谢重编程与肿瘤的发生发展有着密切联系，为肿瘤细胞提供生长优势，帮助其逃避凋亡，同时为肿瘤的转移创造合适的环境，促进肿瘤转移。本文概述了Warburg效应的发生机制及其对肿瘤转移的作用。

Collagen density and alignment in responsive and resistant trastuzumab-treated breast cancer xenografts.

Tumor collagen characteristics influence tumor malignancy, invasion, and metastasis. This study investigates the effects of trastuzumab (Tz) on the collagen of Tz-responsive (BT474) and Tz-resistant (HR6) breast cancer xenografts. Collagen content was assessed by in vivo second harmonic generation (SHG) imaging and histological trichrome staining of tumor sections. Collagen SHG imaging of control BT474 and HR6 tumors demonstrated increased collagen density after 14 days of treatment (p < 0.05). Trichrome staining revealed decreased collagen in Tz-treated BT474 and HR6 tumors at 2, 5, and 14 days of treatment, suggesting that Tz affects the tumor microenvironment independent of epithelial cell response. Additionally, collagen alignment analysis revealed significantly less aligned collagen in the Tz-treated BT474 tumors at day 14 compared with control BT474 tumors. There was no correlation between SHG endpoints (collagen density and alignment) and trichrome staining (p > 0.05), consistent with the physically distinctive nature of these measurements. There was also no correlation between tumor size and collagen endpoints (p > 0.05). These results identify changes within the collagen compartment of the tumor microenvironment following Tz treatment, which are independent from the tumor cell response to Tz, and demonstrate that intravital collagen SHG imaging is capable of measuring dynamic changes in tumor microenvironment following treatment that complements trichrome staining.

Effect of coadministered beta blocker and COX-2 inhibitor to patients with pancreatic cancer prior to receiving albumin-bound (Nab) paclitaxel.

302 Background: Paclitaxel protein-bound particles nab-paclitaxel plus gemcitabine is rapidly becoming the standard of care for patients with metastatic adenocarcinoma of the pancreas (mPCa). Preclinical and clinical studies of beta-blockers and NSAIDs show a benefit of these drugs in pancreatic cancer. The aim of this study was to evaluate the impact of the co-administration of the beta blocker propranolol (P) and the selective COX-2 inhibitor etodolac (E) on survival of patients with mPCa receiving GemNab as standard of care. PE is presumed to target the adrenergic and prostaglandin stress systems activated in cancer that induce changes in tumor microenvironment, immune system, and HPA axis leading to tumor promotion and immune tolerance. Data on the use of nab-paclitaxel plus gemcitabine (GemNab) with a beta blocker and NSAID in the clinical setting is lacking. Methods: Patients with mPCa were eligible for this randomized investigator initiated trial. Patients received PE daily for one week prior to starting GemNab. PE was administered in a chronodosed regimen to maximize the therapeutic benefit and minimize side effects. The primary endpoint was survival. Twenty-three patients were randomized to either GemNab or GemNab after one week of propranolol and etodolac (PEGemNab). The median age was 62.8 years; 68.2% male. Pain at time of diagnosis as reported in 80% of the patients in the GemNab arm and in 76.9% of the PEGemNab arm and jaundice was observed in 40% and 23% of the GemNab and PEGemNab arms, respectively. Results: Progression free survival was 7.2 and 11.8 months in the GemNab and PEGemNab arms, respectively. Overall survival was 10.5 months for the GemNab arm and 15.9 months for the PEGemNab arm. The treatment was well-tolerated with no unexpected adverse events. Conclusions: Administration of PE one week prior to Paclitaxel protein-bound particles with gemcitabine significantly increased progression free and overall survival. No unexpected adverse reactions were seen. Additional data on cytokine and cancer markers will be presented.

Looking out for cancer stem cells' properties: the value-driving role of CD44 for personalized medicines.

The expression of CD44 tags cells with stemness-associated properties (cancer initiating cells or cancer stem cells - CSC). This membrane glycoprotein with a cytoplasmic domain indirectly associated with the cellular cytoskeleton, has a crucial role in tumorigenesis. The CD44 receptor enables the cell to respond to changes in tumor microenvironment, promoting several signaling events related to tumor initiation, progression and fixation in distant host tissues. Although the contribution of this transmembrane protein in gene regulation remains unclear, its overexpression in adenocarcinomas, mostly supported by microRNA (miR)-mediated upregulation of target mRNA, is widely accepted. Herein, we gather the evidence that CD44 is one of the most predominant markers of malignant cells and may be found in diverse phenotypes associated with tumor progression. Additionally, CD44 tumor receptors were found to have different roles at a transcriptional level. Thus, innovative therapeutic strategies should rely heavily on its metastasis-promoting ability. Furthermore, the concept of selectively targeting cell sub-populations may be used to develop specific therapeutic and/or diagnostic systems. An approach based on targeting CD44⁺ cells might provide a strategy to design guided-therapeutic systems against multiple malignant cells including putative CSC.

Overexpression of FGF9 in prostate epithelial cells augments reactive stroma formation and promotes prostate cancer progression.

Bone metastasis is the major cause of morbidity and mortality of prostate cancer (PCa). Fibroblast growth factor 9 (FGF9) has been reported to promote PCa bone metastasis. However, the mechanism by which overexpression of FGF9 promotes PCa progression and metastasis is still unknown. Herein, we report that transgenic mice forced to express FGF9 in prostate epithelial cells (F9TG) developed high grade prostatic intraepithelial neoplasia (PIN) in an expression level- and time-dependent manner. Moreover, FGF9/TRAMP bigenic mice (F9TRAMP) grew advanced PCa earlier and had higher frequencies of metastasis than TRAMP littermates. We observed tumor microenvironmental changes including hypercellularity and hyperproliferation in the stromal compartment of F9TG and F9TRAMP mice. Expression of TGFβ1, a key signaling molecule overexpressed in reactive stroma, was increased in F9TG and F9TRAMP prostates. Both in vivo and in vitro data indicated that FGF9 promoted TGFβ1 expression via increasing cJun-mediated signaling. Moreover, in silico analyses showed that the expression level of FGF9 was positively associated with expression of TGFβ1 and its downstream signaling molecules in human prostate cancers. Collectively, our data demonstrated that overexpressing FGF9 in PCa cells augmented the formation of reactive stroma and promoted PCa initiation and progression.

CS-37 * DUAL FUNCTIONS OF EFEMP1 IN MALIGNANT GLIOMA IN RESPECT TO REGULATION OF EGFR AND NOTCH SIGNALING PATHWAYS

EFEMP1 exerts both tumor suppressive and oncogenic effects in glioma. EFEMP1's binding to EGFR accounts for its suppression of the intracranial (i.c.) tumorigenicity of glioma cells expressing high levels of EGFR. This finding is consistent with an overall favorable prognosis value of EFEMP1 expression in gliomas, specifically high EGFR-expression ones. We also found that in gliomas without EGFR overexpression, heightened levels of EFEMP1 expression were associated with unfavorable patient outcomes in prognosis models. To delineate the cell context-dependent function of EFEMP1 in glioma, we compared the effect of EFEMP1 to EGFR and NOCTH signaling activities in different glioma cell lines/primary cultures and their derived subpopulation lines. The results showed a common effect of EFEMP1 on blocking EGFR signaling, but variations on NOTCH signaling. In U87 glioma cell line, EFEMP1 blocked EGFR while activated NOTCH, with overall suppression of i.c. tumorigenicity. In U251 glioma cell line, EFEMP1 blocked EGFR but no effect on NOTCH signaling, and overall suppression of i.c. tumorigenicity. In U251-derived subculture (U251-NS) which was enriched with stem-like tumor initiating cells where EGFR expression level was low, EFEMP1 up-regulated NOTCH1, HES1, and HEY1 gene expressions. The effect of EFEMP1 to U251-NS i.c. tumorigenicity was contradictory. It changes the effect of tumor promoting to tumor suppressing by increasing inoculum size. With increase of inoculum size in i.c. xenograft model or exposing cells to serum in in vitro culture, there was an increase of chromosome 7 copy score (total chromosome 7 number per cell in xenografts/cell culture) and increase of EGFR expression. EFEMP1 changed pro-invasion function to anti-growth, hence reduced the alteration on equilibrium of tumor heterogeneity in responding to changes of tumor microenvironment. In conclusion, the effect of EFEMP1 up or down-regulation in malignant glioma needs to be studied in respect to the status of EGFR and NOTCH signaling activities.

Tungsten targets the tumor microenvironment to enhance breast cancer metastasis.

The number of individuals exposed to high levels of tungsten is increasing, yet there is limited knowledge of the potential human health risks. Recently, a cohort of breast cancer patients was left with tungsten in their breasts following testing of a tungsten-based shield during intraoperative radiotherapy. While monitoring tungsten levels in the blood and urine of these patients, we utilized the 66Cl4 cell model, in vitro and in mice to study the effects of tungsten exposure on mammary tumor growth and metastasis. We still detect tungsten in the urine of patients' years after surgery (mean urinary tungsten concentration at least 20 months post-surgery = 1.76 ng/ml), even in those who have opted for mastectomy, indicating that tungsten does not remain in the breast. In addition, standard chelation therapy was ineffective at mobilizing tungsten. In the mouse model, tungsten slightly delayed primary tumor growth, but significantly enhanced lung metastasis. In vitro, tungsten did not enhance 66Cl4 proliferation or invasion, suggesting that tungsten was not directly acting on 66Cl4 primary tumor cells to enhance invasion. In contrast, tungsten changed the tumor microenvironment, enhancing parameters known to be important for cell invasion and metastasis including activated fibroblasts, matrix metalloproteinases, and myeloid-derived suppressor cells. We show, for the first time, that tungsten enhances metastasis in an animal model of breast cancer by targeting the microenvironment. Importantly, all these tumor microenvironmental changes are associated with a poor prognosis in humans.

Tumor microenvironmental changes induced by the sulfamate carbonic anhydrase IX inhibitor S4 in a laryngeal tumor model.

Background and PurposeCarbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3′(3″,5″-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding.MethodsSCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR.ResultsCAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed.ConclusionAs the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.

Differential expression of stress and immune response pathway transcripts and miRNAs in normal human endothelial cells subjected to fractionated or single-dose radiation.

Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy.

Benzyl isothiocyanate suppresses high-fat diet-stimulated mammary tumor progression via the alteration of tumor microenvironments in obesity-resistant BALB/c mice.

We previously reported that a high-fat diet (HFD) and M2-macrophages induce changes in tumor microenvironments and stimulate tumor growth and metastasis of 4T1 mammary cancer cells in BALB/c mice. In this study, we attempted to determine whether benzyl isothiocyanate (BITC) inhibits HFD-induced changes in tumor progression and in tumor microenvironments. Four groups of female BALB/c mice (4-week-old) were fed on a control diet (CD, 10 kcal% fat) and HFD (60 kcal% fat) containing BITC (0, 25, or 100 mg/kg diet) for 20 weeks. Following 16 weeks of feeding, 4T1 cells (5×10(4) cells) were injected into the mammary fat pads, and animals were killed 30 d after the injection. HFD feeding increased solid tumor growth and the number of tumor nodules in the lung and liver, as compared to the CD group, and these increases were inhibited by BITC supplementation. The number of lipid vacuoles, CD45+ leukocytes and CD206+ M2-macrophages, expression of Ki67, levels of cytokines/chemokines, including macrophage-colony stimulating factor (M-CSF) and monocyte chemoattractant protein-1, and mRNA levels of F4/80, CD86, Ym1, CD163, CCR2, and M-CSF receptor were increased in the tumor tissues of HFD-fed mice, and these increases were inhibited by BITC supplementation. In vitro culture results demonstrated that BITC inhibited macrophage migration as well as lipid droplet accumulation in 3T3-L1 cells. These results suggest that suppression of lipid accumulation and macrophage infiltration in tumor tissues may be one of the mechanisms by which BITC suppresses tumor progression in HFD-fed mice.

Changes in tumor microenvironments in high‐fat diet‐fed mice: role of macrophages and adipocytes (37.5)

Using several mouse tumor models, we have shown that the chronic consumption of a high‐fat diet (HFD) stimulates solid tumor growth and metastasis and thereby reduces the survival rate. The present study examined the mechanisms by which HFD stimulates tumor promotion. Angiogenesis and lymphangiogenesis in tumor tissues and lymph node (LN)s are markedly increased in HFD‐fed mice. HFD feeding increases adipocytes and M2‐macrophage (M2‐MΦ)s in tumor tissues and M2‐MΦs in adipose tissues of tumor‐bearing mice. Results from in vivo and in vitro co‐culture studies revealed that the crosstalk between adipocytes, tumor cells, M2‐MΦs, and/or lymphatic endothelial cell (LEC)s stimulates the production of several factors which stimulate tumor cell/monocyte migration, angiogenesis, and lymphangiogenesis via endocrine and paracrine mechanisms. In HFD‐fed mice, increased chemokine ligands in LECs recruit tumor cells expressing high levels of their receptors, thereby increasing LN metastasis. Our results indicate that increased adipocytes in HFD‐fed mice play important roles in the recruitment of M2‐MΦs in tumor tissues and lymphangiogenesis as well as tumor cell migration, thereby stimulating tumor metastasis.

Both Intermittent and Continuous Dosing Regimens Of Pomalidomide Mediate Broad Activation Of Innate and Adaptive Immunity In Relapsed/Refractory Myeloma: Results Of A Phase II Randomized Trial

Pomalidomide (POM) is a novel IMiD® immunomodulatory agent with clinical activity in several settings including in relapsed / resistant myeloma (RRMM). Several preclinical studies have documented the immunologic effects of IMiD® immunomodulatory drugs. IMiD® now form the backbone of several emerging combination therapies in hematologic malignancies. In prior studies, the clinical activity of POM in relapsed myeloma has been demonstrated using both continuous and intermittent dosing regimens with and without steroids. However the impact of the specific POM dosing regimen and the effect of concurrent steroids (as commonly utilized in most combination regimens) on POM-induced immune activation in vivo is unknown. In order to evaluate these issues more directly, we analyzed samples from patients enrolled in a randomized phase II clinical trial comparing two POM dosing schedules. Comparison of drug-induced immune activation between the two dosing schedules was one of the pre-specified endpoints in this study. Patients (n=39) with RRMM documented to be refractory to lenalidomide were randomized to therapy with POM 2 mg/day for 28/28 days (Arm A, n=19) or POM 4 mg/day for 21/28 days (Arm B, n=20) of a 28 day cycle. All patients (pts) received POM alone for cycle 1, followed by the addition of dexamethasone (DEX) at 40 mg weekly in subsequent cycles in both arms. In recent studies, we have shown that immune effects of lenalidomide are manifest early, within a week of initiation of therapy (Richter et al, Blood 121:423, 2013). Therefore each patient was analyzed 1 week after initiation of POM alone at 2 or 4 mg (cycle 1) or POM + DEX (cycle 2). POM therapy led to an increase in T cells at day 7 after initiation of therapy (mean percent increase compared to baseline 47%, p <0.01). This POM-induced T cell expansion consisted predominantly of CD8+ T cells (mean increase compared to baseline 40%, p<0.01). POM therapy also led to an increase in circulating natural killer (NK) cells (mean increase compared to baseline 92%, p<0.01) which peaked at 7 days post-initiation of therapy. POM-induced NK expansion was associated with an increase in the expression of NKG2D as well as CD16 and CD56, consistent with NK activation in vivo. Changes in T and NK cells following cycle 2 (POM+DEX) were compared with the data on cycle 1 (POM alone) in the same patient to evaluate the impact of DEX. Addition of DEX led to dampening of drug-induced T cell activation in vivo, but surprisingly did not dampen drug induced NK activation. Comparison of data from the two cohorts demonstrated that both 2 mg and 4 mg dose of POM led to comparable degree of immune activation in vivo, particularly for NK cells. In selected patients, post-treatment bone marrow samples were available after completion of at least 2 cycles of therapy. Comparison of the transcriptome of the CD138-depleted fraction from these marrows (representing bulk tumor microenvironment) identified nearly 300 genes upregulated in the bone marrow microenvironment post therapy compared to paired baseline samples. Interestingly, these genes were highly enriched in genes associated with T and NK activation. As POM led to activation of both innate and adaptive immunity, we also analyzed drug-induced changes in the myeloid compartment. Comparison of gene expression profiles (GEP) of purified CD14+ monocytes before and after POM indicated that POM therapy leads to a distinct GEP signature in myeloid cells in vivo. Finally, correlation of drug-induced changes in immune cells with clinical response demonstrated that an increase in post-therapy T cells correlated with clinical response, but post-treatment increase in NK cells did not. Together these data demonstrate that both 2 mg and 4 mg dose of POM can mediate broad activation of both innate and adaptive immunity in vivo, even in the setting of immune paresis associated with advanced, heavily pre-treated disease. Both continuous and intermittent dosing schedules of POM have comparable pharmacodynamic effects on immune activation. POM-induced immune activation is detected even with concurrent steroids and immune-related genes constitute a dominant component of drug induced changes in tumor microenvironment. POM-mediated immune activation may thus be exploited in the context of combination with immune therapies as well as monoclonal antibodies. Disclosures:No relevant conflicts of interest to declare.

Epigenetics and cancer metabolism

Cancer cells adapt their metabolism to support proliferation and survival. A hallmark of cancer, this alteration is characterized by dysfunctional metabolic enzymes, changes in nutrient availability, tumor microenvironment and oncogenic mutations. Metabolic rewiring in cancer is tightly connected to changes at the epigenetic level. Enzymes that mediate epigenetic status of cells catalyze posttranslational modifications of DNA and histones and influence metabolic gene expression. These enzymes require metabolites that are used as cofactors and substrates to carry out reactions. This interaction of epigenetics and metabolism constitutes a new avenue of cancer biology and could lead to new insights for the development of anti-cancer therapeutics.