Reduction In Primary Tumor Growth Research Articles

Abstract 3925: Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity

Abstract Despite early diagnosis, 10-20% of breast cancer patients experience metastatic disease within 5-10 years. While it is established that breast cancer cells have the capacity to evade immune responses, the mechanisms driving this evasion remain elusive. Therefore, finding ways to enhance anti-tumor immunity is crucial to improve patient outcomes. Dickkopf-1 (DKK1) is a Wnt inhibitor whose levels correlate with poor prognosis and decreased immune infiltration in various cancers. In this study, we used mice orthotopically injected with the PyMT, EO771, and 4T1 breast cancer lines. In all models, we found elevated DKK1 serum levels, and its neutralization with an αDKK1 Ab resulted in a significant reduction in primary tumor growth but also in metastatic dissemination. To identify the source of DKK1, we examined its expression in the tumor. DKK1 was detected in stromal cells with a fibroblast-like morphology but not in cancer cells or immune infiltrates. Co-staining confirmed colocalization between DKK1 and the CAF marker αSMA. To address the role of CAF-derived DKK1, we specifically deleted DKK1 in CAFs by generating FSP1CreDkk1fl/fl and αSMACreERT2Dkk1fl/fl mice. Both models showed a significant reduction in primary tumor growth while maintaining comparable serum DKK1 levels, indicating locally produced DKK1 supports tumor growth. To understand how DKK1 promotes tumor progression, we conducted bulk RNAseq of tumor cells isolated from IgG or αDKK1 treated mice. While no changes related to cell viability or cell cycle were observed, immune response-related genes were upregulated in αDKK1-treated cells. In support of this, αDKK1 did not reduce tumor growth in NSG immune-compromised mice. To pinpoint the immune cell targeted by DKK1, we depleted T, NK cells, or macrophages in WT tumor-bearing mice treated with αDKK1. Remarkably, only NK cell depletion negated αDKK1 anti-tumor effects. In vitro assays further demonstrated that rDKK1 or the presence of CAFs reduced NK cell cytotoxicity against tumor cells while αDKK1 restored it. To assess whether DKK1 levels correlated with tumor progression and immune suppression in breast cancer patients, we analyzed DKK1 serum levels and circulating NK cells at the diagnosis of metastatic bone disease and 15-18 months after standard care. DKK1 levels were significantly elevated in patients with progressive bone disease compared to those with stable disease. Importantly, NK cells from the progressive disease cohort had reduced perforin and granzyme B expression at the follow-up visit compared to baseline, indicating reduced cytotoxicity. Furthermore, rDKK1 reduced perforin in NK cells from healthy donors. In sum, our data show DKK1 is an important regulator of breast cancer progression, and we report for the first time DKK1 directly impairs NK cell cytotoxicity, creating an immune suppressive environment resistant to treatment. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3925.

Abstract 3349: Identification of new potential vulnerabilities for differentiation-based therapeutic strategies against osteosarcoma

Abstract Osteosarcoma (OS), a mesenchymal bone tumor affecting mainly children and adolescents is characterized by a particularly aggressive behavior, with 20% of patients showing lung metastasis already at diagnosis. Current OS treatment strategies rely on multi-drug chemotherapy, and the prognosis for metastatic cases remains grim, underscoring the pressing need for novel therapeutic approaches. OS is considered a differentiation-related disorder, stemming from the inability of mesenchymal stem cells or osteoblastic progenitors to progress towards terminal differentiation. In our study we explored the impact of targeting ZEB1, a transcription factor known to play a pivotal role in maintaining mesenchymal and stemness characteristics in cancer, on OS cells. We employed CRISPR-Cas9 and shRNA interference techniques to inhibit ZEB1 expression in mouse immune competent OS models. In vitro, ZEB1 deficiency significantly diminished the stemness potential of OS cells, as assessed by spheroid formation assays, while promoting osteogenic differentiation. In vivo, ZEB1 deletion in OS cells led to a significant reduction in primary tumor growth. Tumors originating from ZEB1 KO cells exhibited a more differentiated morphology and higher extracellular matrix deposition compared to controls. Intriguingly, the absence of ZEB1 in tumor cells also influenced the immune infiltrate, with ZEB1-deficient tumors showing significantly fewer pro-tumoral CD206+ M2-like macrophages. This observation is in line with the significant down-modulation of CCL2 expression we detected in ZEB1 KO cells. When injected intravenously, ZEB1-deficient cells produced fewer and smaller lung metastases than control cells. Transcriptional changes induced by ZEB1 deletion were investigated by gene expression profile (GEP) and identified 849 down-regulated and 1093 up-regulated genes in ZEB1 knockout (KO) clones versus ZEB1-competent controls. Gene set enrichment analysis showed a down-modulation of pathways related to cellular proliferation and survival, such as MTORC1 signaling, MYC targets, GM2 checkpoint, E2F targets and oxidative phosphorylation in the absence of ZEB1. Single gene analysis identified among the most up-regulated genes in ZEB1-deficient cells Sfrp1, a negative regulator of the WNT/b-CAT pathway, which in turn regulates MYC, whose pathway is indeed repressed in ZEB1 KO cells. We hypothesize that a high expression of SFRP1 could vicariate the effects of ZEB1 inhibition on OS cell stemness, differentiation state, and in vivo aggressiveness, and therefore could act as a tumor suppressor, which could be potentially exploited for therapeutic strategies. Citation Format: Caterina Cascini, Daniele Lecis, Valeria Cancila, Katia Scotlandi, Claudio Tripodo, Mario P. Colombo, Claudia Chiodoni. Identification of new potential vulnerabilities for differentiation-based therapeutic strategies against osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3349.

Abstract 3182: Role of Frizzled 7 in melanoma development and metastasis

Abstract The frizzled (Fzd) family of proteins are 7-transmembrane G-protein coupled receptors (GPCRs) that transduce signals mainly for Wnt ligands. There are 10 Fzds identified in mammals, and they are involved in multiple cellular and biological functions via signaling through the canonical Wnt/beta-catenin, planar cell polarity, and calcium signaling pathways. Our previous work revealed a pro-metastasis role of Fzd6 in melanoma. This project focuses on Fzd7, a homolog of Fzd6, in melanoma. We found that FZD7 is highly expressed in multiple melanoma cell lines. siRNA knockdown of FZD7 does not affect cell proliferation but significantly reduces cell invasion in A375, Hs294T, and SK-MEL28 cells in vitro. To determine the in vivo role of Fzd7 in melanoma, we generated Fzd7 knockout YUMM1.7 cell lines for xenograft experiments using CRISPR. All three Fzd7 knockout clones showed a significant reduction in primary tumor growth when xenografted in C57BL/6 mice. To determine the potential mechanism of Fzd7 in melanoma growth and invasion, we performed bulk RNA-Seq on the YUMM1.7 knockout cell lines and identified 215 downregulated and 31 upregulated genes (with a fold change of 2, p<0.05). We are in the process of confirming these transcriptional profile changes. Further functional analysis will be performed using gain- and loss-of-function approaches to determine the downstream mechanisms of Fzd7 in melanoma growth and invasion. Citation Format: Minakshi Nihal, Sheikh A. Umar, An-Bo A. Chang, Nihal Ahmad, Hao Chang. Role of Frizzled 7 in melanoma development and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3182.

Direct In Vivo Activation of T Cells with Nanosized Immunofilaments Inhibits Tumor Growth and Metastasis.

Adoptive T cell therapy has successfully been implemented for the treatment of cancer. Nevertheless, ex vivo expansion of T cells by artificial antigen-presenting cells (aAPCs) remains cumbersome and can compromise T cell functionality, thereby limiting their therapeutic potential. We propose a radically different approach aimed at direct expansion of T cells in vivo, thereby omitting the need for large-scale ex vivo T cell production. We engineered nanosized immunofilaments (IFs), with a soluble semiflexible polyisocyanopeptide backbone that presents peptide-loaded major histocompatibility complexes and costimulatory molecules multivalently. IFs readily activated and expanded antigen-specific T cells like natural APCs, as evidenced by transcriptomic analyses of T cells. Upon intravenous injection, IFs reach the spleen and lymph nodes and induce antigen-specific T cell responses in vivo. Moreover, IFs display strong antitumor efficacy resulting in inhibition of the formation of melanoma metastases and reduction of primary tumor growth in synergy with immune checkpoint blockade. In conclusion, nanosized IFs represent a powerful modular platform for direct activation and expansion of antigen-specific T cells in vivo, which can greatly contribute to cancer immunotherapy.

Effect of dual properties of regorafenib in osteosarcoma on tumor progression and bone parameters in mouse preclinical models.

e23501 Background: Osteosarcoma (OS) constitutes the most frequent malignant primary bone tumor in children and young adults. Actual treatments in OS are based in a multimodal therapy that combines pre- and post-surgery polychemotherapies including cisplatin, doxorubicin, ifosfamide and/or methotrexate. Regardless the use of this polychemotherapeutic approach, the five years overall survival for patients has not improved during the last four decades and there is a clinical need to identify new active drugs in OS. An interesting strategy implies the use of compounds that showed therapeutic benefits in other solid tumors. Regorafenib, a small molecule with multi-target tyrosine kinase inhibitory activity against FGFR-1 and -2, KIT, PDGFR, RAF, RET, TIE2 and VEGFR-1, -2 and -3, showed an anti-angiogenic/oncogenic properties in various solid tumors that includes metastatic colorectal cancer, hepatocarcinoma and breast cancer. In addition, regorafenib also inhibits in vitro the proliferation of several pediatric solid tumor cell lines such as rhabdomyosarcoma, Ewing sarcoma and OS. Methods: The present work aims to decipher the in vitro and in vivo effects of regorafenib in OS preclinical models. Results: The treatment of MNNG-HOS, KHOS and 143B osteosarcoma cell lines with Regorafenib resulted in the inhibition of cell proliferation in a dose dependent manner. The gene expression profiles of MNNG-HOS and 143B cells treated or not with regorafenib and analyzed by NanoString technology revealed a specific regulation of molecular pathways by regorafenib in both cell lines. The in vivo effect of regorafenib was then analyzed in immunocompetent (mouse MOS-J cells inoculated in C57Bl/6 mice) and immunodeficient (human MNNG-HOS and 143B OS cells injected in Nude mice) murine osteosarcoma models. In all the cases, a significant reduction of primary tumor growth was observed as well as a significant decrease of lung metastases compared to untreated mice. Interestingly, the cortical and trabecular bone mineral density were significantly increased in regorafenib treated mice compared to untreated groups as demonstrated by X ray microtomodensitometry in all models. Conclusions: Our results support the use of regorafenib as an alternative treatment in metastatic OS and demonstrated for the first time the its bone protective effect.

Abstract 662: Local and systemic effects of Dickkopf-1 during breast cancer progression by limiting NK cell-mediated killing

Abstract While most breast cancer patients are diagnosed early, approximately 10-20% recur with the metastatic disease within 10 years. Unfortunately, cures are limited once recur, especially if the tumor spreads to the bone, a site resistant to therapies. Elevated levels of the Wnt inhibitor dickkopf-1 (Dkk1) are detected in breast cancer patients and correlate with the progression of bone metastases. We also find that breast cancer patients with progressive metastatic bone disease unresponsive to standard-of-care therapies have higher Dkk1 levels than patients with stable disease. Intriguingly, serum Dkk1 levels do not always correlate with Dkk1 expression in cancer cells. Thus, it is important to understand the source and role of Dkk1 in breast cancer. In this study, we used mice orthotopically injected with PyMT, EO771, and 4T1 breast cancer lines. Although Dkk1 was not expressed in the tumor cells, Dkk1 serum levels were upregulated in all models. To determine the role of Dkk1 in tumor progression, we used a Dkk1 neutralizing antibody (α-Dkk1) and found a significant reduction in primary tumor growth and in the bone of mice intratibially injected with PyMT tumor cells compared to IgG. To understand the source of Dkk1, we isolated the tumor mass and the bones from tumor-bearing mice. Dkk1 was highly expressed by the osteoblasts (OBs) in the bone and to a less extent by the cancer-associated fibroblasts (CAFs) in the tumor stroma. To address the role of bone- versus CAF-derived Dkk1, we generated mice with specific deletion of Dkk1 in the OBs (OsxCre;Dkk1fl/fl), or CAFs (Fsp1Cre;Dkk1fl/fl and aSMACreERT2;Dkk1fl/fl). To our surprise, all our mouse models showed a significant reduction in primary tumor growth, suggesting systemic and local effects of Dkk1. Further supporting direct local effects of Dkk1, WT mice co-injected with PyMT and Dkk1 deficient CAFs showed reduced tumor growth compared to mice co-injected with Dkk1 sufficient CAFs. To understand how Dkk1 exerts its pro-tumorigenic effects, we performed bulk RNAseq of tumor cells isolated from IgG and α-Dkk1 treated mice. To our surprise, we did not detect changes in pathways related to cell proliferation, survival, apoptosis, and cytoskeletal reorganization. However, pathways related to immune responses were upregulated in the α-Dkk1 treated tumors. Indeed, Dkk1 neutralization did not reduce tumor growth in the NSG immune-compromised mice. To investigate which immune population was targeted by Dkk1, we treated tumor-bearing mice with α-Dkk1 in combination with depletion of T, NK cells, or macrophages. Surprisingly, only NK cell depletion abrogated α-Dkk1 anti-tumor effects. In vitro assays further confirmed reduced NK cell killing efficiency against PyMT tumor cells in the presence of recombinant Dkk1. Our data show that Dkk1 exerts local and systemic effects to promote tumor progression by modulating the killing efficiency of NK cells. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Local and systemic effects of Dickkopf-1 during breast cancer progression by limiting NK cell-mediated killing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 662.

Parsing β-catenin’s cell adhesion and Wnt signaling functions in malignant mammary tumor progression

During malignant progression, epithelial cancer cells dissolve their cell-cell adhesion and gain invasive features. By virtue of its dual function, β-catenin contributes to cadherin-mediated cell-cell adhesion, and it determines the transcriptional output of Wnt signaling: via its N terminus, it recruits the signaling coactivators Bcl9 and Pygopus, and via the C terminus, it interacts with the general transcriptional machinery. This duality confounds the simple loss-of-function analysis of Wnt signaling in cancer progression. In many cancer types including breast cancer, the functional contribution of β-catenin's transcriptional activities, as compared to its adhesion functions, to tumor progression has remained elusive. Employing the mouse mammary tumor virus (MMTV)-PyMT mouse model of metastatic breast cancer, we compared the complete elimination of β-catenin with the specific ablation of its signaling outputs in mammary tumor cells. Notably, the complete lack of β-catenin resulted in massive apoptosis of mammary tumor cells. In contrast, the loss of β-catenin's transcriptional activity resulted in a reduction of primary tumor growth, tumor invasion, and metastasis formation in vivo. These phenotypic changes were reflected by stalled cell cycle progression and diminished epithelial-mesenchymal transition (EMT) and cell migration of breast cancer cells in vitro. Transcriptome analysis revealed subsets of genes which were specifically regulated by β-catenin's transcriptional activities upon stimulation with Wnt3a or during TGF-β-induced EMT. Our results uncouple the signaling from the adhesion function of β-catenin and underline the importance of Wnt/β-catenin-dependent transcription in malignant tumor progression of breast cancer.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

A systematic review on the efficacy and safety of low molecular weight heparin as an anticancer therapeutic in preclinical animal models

ObjectiveThe therapeutic effects of low molecular weight heparins (LMWH) may extend past thrombosis prevention, with preclinical evidence demonstrating anti-metastatic properties. Clinical evidence on the topic, however, remains controversial. A systematic review of preclinical evidence may help elucidate reasons for this contradictory evidence. The objective of our systematic review is to assess the anti-metastatic properties of LMWHs in solid tumour animal models. MethodsMEDLINE, Embase, Web of Science and PubMed were searched from inception to May 12th, 2020. All articles were screened independently and in duplicate. Studies that compared LMWH to a placebo or no treatment arm in solid tumour animal models were included. The primary outcome was the burden of metastasis. Secondary outcomes included primary tumour growth and mortality. The risk of bias was assessed in duplicate using a modified Cochrane Risk of Bias tool. ResultsForty-two studies were included in the review. Administration of a LMWH was associated with a significant decrease in the burden of metastasis (SMD −2.18; 95% CI −2.66 to −1.70). Additionally, the administration of a LMWH was also associated with a significant reduction in primary tumour growth (SMD −1.95; 95% CI −2.56 to −1.34) and risk of death (RR 0.39; 95% CI 0.16–0.97). All included studies were deemed to be at an unclear risk of bias for at least one methodological criterion. ConclusionsOur results demonstrate that LMWH can effectively reduce metastatic burden and reduce tumour growth in preclinical animal models of solid tumour malignancies. Reasons for the contradiction with clinical evidence require further exploration.

Delivery of Immunotherapeutic Nanoparticles to Tumors via Enzyme-Directed Assembly.

Amphiphilic diblock copolymers are prepared by ring opening metathesis polymerization, with one block containing hydrophobic Toll-like receptor 7 (TLR7) agonists and one block containing hydrophilic peptides as substrates for matrix metalloproteinases (MMPs). A fluorescent label is incorporated into the polymer chains for in vivo imaging. Upon dialysis against aqueous solution, polymers form 15 nm spherical micelles. Subsequent exposure to MMP-9 elicits a morphological change to yield immunostimulatory microscale assemblies. The intravenous (IV) administration of the formulation to mice bearing 4T1 breast cancer tumors results in nanoparticle accumulation in tumors, reduction in primary tumor growth, and inhibition of lung metastases, as compared to saline-treated animals. Mice administered the parent immunotherapeutic small molecule (1V209) experience significantly increased plasma levels of proinflammatory cytokines IL-6, IP-10, and MCP-1 at 2 h following IV administration, whereas the nanomaterial shows no increase over saline-treated controls. These data suggest that covalently packaging low molecular weight immunotherapeutics at high weight percent loadings in enzyme-responsive nanoparticles maintains drug efficacy while decreasing immunotoxicity, providing a platform for cancer immunotherapeutic delivery.

Abstract 586: CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival

Abstract The 4T1.2 syngeneic murine breast cancer model is an aggressive model of stage IV triple-negative breast cancer. Despite its significant strength, the 4T1.2 model is poorly immunogenic with few defined tumor antigens. The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter for bioluminescence imaging. However, studies have shown that the luciferase (Luc) may serve as a tumor antigen and induce CD8+ cytotoxic T cell responses. The goal of the current study was to determine if luciferase expression in 4T1.2 tumor cells would enhance the immunogenicity of the tumor, and explore the role of immune effector cells in the parental 4T1.2 and luciferase-transfected 4T1.2luc tumor model. In vitro proliferation of the 4T1.2 and 4T1.2luc cells was assessed using the MTS cell proliferation assay. For in vivo experiments, female BALB/c mice were randomized into 2 groups and orthotopically injected with 5x104 4T1.2 or 4T1.2luc cells, respectively. Tumor growth was assessed until day 35 post tumor implantation. Each group was further randomized into 5 subgroups and received i.p. injections of PBS, rat IgG2b mAb, anti-CD4 mAb, anti-CD8 mAb, or anti-asialo GM1. Mice were sacrificed between day 28-35 post tumor implantation, and lung metastases were analyzed for each group. To further evaluate the immunogenicity of luciferase in the 4T1.2luc model, a separate cohort of mice were orthotopically injected with 5x104 4T1.2luc cells and sacrificed at day 35 post tumor implantation. Splenocytes were harvested and cultured in the presence of 1 or 10 µg/ml peptides representing MHC I-restricted Luc epitopes for 5 days, and cytotoxicity of the effector cells was assessed by the chromium-release assay. In vitro proliferation rates were not significantly different between 4T1.2 and 4T1.2luc tumor cells. However, 4T1.2luc tumor-bearing mice showed a significant reduction in primary tumor growth compared to 4T1.2 tumor-bearing mice. 4T1.2 primary tumor growth was not altered by the depletion of any immune cell type, while 4T1.2luc primary tumor growth was significantly increased by the depletion of CD8+ T cells, and reduced by the depletion of NK cells. Depletion of CD4+ T cells, CD8+ T cells or NK cells in 4T1.2luc tumor-bearing mice resulted in a significant reduction in median survival. Splenocytes from 4T1.2luc tumor bearing mice demonstrated Luc-specific cytotoxicity upon ex vivo restimulation with Luc peptides, and a stronger response was observed when cells were restimulated with 1 µg/ml compared to 10 µg/ml Luc peptides. These findings suggest that luciferase may serve as a tumor antigen in the 4T1.2luc tumor model, and host immune components (CD4+ and CD8+ T cells) may play a role in controlling 4T1.2luc primary tumor growth and survival. The stronger immunogenicity of 4T1.2luc conferred by luciferase will enable future immunotherapeutic research that ultimately may benefit breast cancer patients. Citation Format: Yitong Xu, Shizhao Duan, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival [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 586.

The FAK inhibitor BI 853520 exerts anti-tumor effects in breast cancer

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that regulates a plethora of downstream signaling pathways essential for cell migration, proliferation and death, processes that are exploited by cancer cells during malignant progression. These well-established tumorigenic activities, together with its high expression and activity in different cancer types, highlight FAK as an attractive target for cancer therapy. We have assessed and characterized the therapeutic potential and the biological effects of BI 853520, a novel small chemical inhibitor of FAK, in several preclinical mouse models of breast cancer. Treatment with BI 853520 elicits a significant reduction in primary tumor growth caused by an anti-proliferative activity by BI 853520. In contrast, BI 853520 exerts effects with varying degrees of robustness on the different stages of the metastatic cascade. Together, the data demonstrate that the repression of FAK activity by the specific FAK inhibitor BI 853520 offers a promising anti-proliferative approach for cancer therapy.

Abstract 2240: Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces tumor-infiltrating MDSCs expressing PD-L1 and IDO

Abstract Obesity and physical inactivity increase breast cancer risk, while the prevention of weight gain by diet and exercise can be protective. Numerous biological mechanisms are proposed to explain the beneficial effects of weight maintenance, including changes in metabolic, inflammatory and immune mediators. We have previously shown that diet and exercise-induced weight maintenance (WM), in combination with a whole 4T1.2luc tumor cell vaccine (VAX), significantly reduced mammary tumor growth and metastases in the 4T1.2luc murine mammary tumor model. This reduction in growth and metastases occurred concomitantly with an elevation in tumor antigen-induced splenic IFN-γ production and a reduction in the accumulation of splenic myeloid-derived suppressor cells (MDSCs) at day 35 post-tumor implantation. The goal of the current study was to determine if WM+VAX alters the tumor microenvironment at an early stage of tumor growth (day 24 post-tumor implantation) which may contribute to reduced tumor growth and enhanced immune outcomes at day 35 post-tumor implantation. Female BALB/c mice were randomized into sedentary, weight gain (WG) or exercising (access to voluntary running wheel), weight maintenance (WM) groups (n=22-27/group). After 8 weeks on the intervention, all mice were orthotopically injected with 5x104 4T1.2luc cells into the fourth mammary fat pad and continued on their intervention. Once injected, both WG and WM mice were further randomized into vaccination (VAX) or vehicle control (VEH) groups (n=10-15/group) and administered 1x106 irradiated 4T1.2luc cells (VAX) or HBSS (VEH) at day 7, 14 and 21 post-tumor implantation. Mice were sacrificed at day 24 post-tumor implantation and tumor-infiltrating immune cells were isolated for analyses. Both WM+VEH and WM+VAX groups showed a significant reduction in primary tumor growth and splenomegaly compared to both WG groups (p<0.001). Flow cytometric analysis demonstrated that the combination of WM+VAX significantly reduced total tumor-infiltrating MDSCs and MDSCs expressing PD-L1 and indoleamine 2,3-dioxygenase (IDO) compared to WG+VEH (p<0.05). This finding was consistent with a 1.5-4.5 fold decrease in the gene expression level of Pdcd1, Ido1 and Ifng in the total tumor infiltrates. These results suggest that the combination of weight maintenance and the whole tumor cell vaccine may be altering both the number and immunosuppressive capacity of tumor-infiltrating MDSCs. Thus, preventing weight gain through diet and exercise may be an important recommendation in combination with immune-based therapies to enhance efficacy and improve clinical outcomes. This work is supported by R21 CA209144; T32GM108563. W.J.T is currently funded by the NIH T32DK062710 grant; NORC, UAB, Birmngham, AL. Citation Format: Yitong Xu, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. Diet and exercise-induced weight maintenance, alone and in combination with a whole tumor cell vaccine, delays mammary tumor growth and reduces tumor-infiltrating MDSCs expressing PD-L1 and IDO [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2240.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

Targeting of heme oxygenase-1 as a novel immune regulator of neuroblastoma.

Heme oxygenase (HO)-1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO-1 expression in samples from NB patients and show that targeting of HO-1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO-1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO-1 by systemic as well as tumor-specific zinc protoporphyrin-mediated HO-1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO-1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T-effector cells. This in turn reverted HO-1-dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO-1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model.

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC.

Abstract LB-B13: The application of high-dose low fraction image-guided irradiation (IGMI) in combination with anti-CTLA4 immunotherapy results in an additive inhibition of tumor growth in syngeneic tumor models

Abstract INTRODUCTION: Radiotherapy (RT) is used across multiple cancer types in a number of clinical settings. The primary mechanism of action is thought to be through irreversible damage to tumor cell or associated stromal DNA, but there is an increasing body of evidence that irradiation also mobilizes tumor-specific immunity. Recent advances in RT with stereotactic and image-guided micro-irradiation (IGMI) have resulted in an increase in tumor specific targeting with a corresponding reduction in associated side-effects. The resultant use of high-dose, reduced fraction dosing regimens (hypofractionation) has resulted in improved clinical response. However, IGMI in the preclinical setting is less common and typically studies utilize whole body irradiation with lead shielding or simple single beam techniques to focus the radiation. Here we report the application of high-dose, low fraction IGMI to demonstrate an additive combination outcome of radiotherapy and immunotherapy and an abscopal effect in a preclinical model. EXPERIMENTAL PROCEDURES: CT26 mouse colon and bioluminescent 4T1 mammary carcinoma cells were implanted subcutaneously into BALB/c mice. Subcutaneous tumor growth was monitored by caliper measurement and bioluminescent imaging (BLI) was carried out to confirm metastatic 4T1 lung tumors. Tumors (∼100-200mm3) were treated with hypofractionated IGMI, anti-CTLA4 therapy or a combination of both. For IGMI, mice were anaesthetized and CBCT images were acquired using the small animal research platform (SARRP; Xstrahl Ltd). Fractionated irradiation (5 × 6Gy) was administered to the isocenter of the tumor using a multi beam approach to spare the surrounding tissue. Bodyweight and clinical signs were monitored daily. At termination the primary and metastatic lung (4T1) tumors were collected and assessed for immune cell infiltration by FACs. RESULTS: Response to the hypofractionated IGMI regimen and anti-CTLA4 either alone or in combination was well tolerated with no adverse effects or significant body weight-loss. Monotherapy with anti-CTLA4 exerted no statistically significant effect on primary or metastatic (4T1) tumor growth and treatment with RT resulted in a modest primary tumor growth inhibition (TGI; 40%). When the two treatments were combined, TGI increased (60%) which was statistically significant compared to RT monotherapy, indicating an additive effect. Additionally, there was a decrease in normalized lung cell counts following combination treatment (4T1) indicating an abscopal response on the lung metastases. This decrease in lung tumor burden corresponded to an increase in both the CD8+ T-cells and the ratio of Teff:Treg in the lung samples. CONCLUSIONS: Hypofractionated IGMI when combined with anti-CTLA4 treatment had minimal side effects and showed statistically significant additive reduction in primary tumor growth. Furthermore, combination treatment resulted in a reduced metastatic lung burden indicating an abscopal effect that appears to be driven by an increase in CD8+ T-cells. The SARRP platform enables clinically relevant radiotherapy regimens to preclinical models facilitating improved evaluation of combinations strategies with immunotherapeutics and progression to the clinic. Citation Format: Andrew Mckenzie, Nektaria Papadopoulou, Yinfei Yin, Shonna Glenn, Russell Garland, Neil Williams, Qian Shi, Rajendra Kumari. The application of high-dose low fraction image-guided irradiation (IGMI) in combination with anti-CTLA4 immunotherapy results in an additive inhibition of tumor growth in syngeneic tumor models. [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 LB-B13.

Long Noncoding RNA Ceruloplasmin Promotes Cancer Growth by Altering Glycolysis.

Long noncoding RNAs (lncRNAs) significantly influence the development and regulation of genome expression in cells. Here, we demonstrate the role of lncRNA ceruloplasmin (NRCP) in cancer metabolism and elucidate functional effects leading to increased tumor progression. NRCP was highly upregulated in ovarian tumors, and knockdown of NRCP resulted in significantly increased apoptosis, decreased cell proliferation, and decreased glycolysis compared with control cancer cells. In an orthotopic mouse model of ovarian cancer, siNRCP delivered via a liposomal carrier significantly reduced tumor growth compared with control treatment. We identified NRCP as an intermediate binding partner between STAT1 and RNA polymerase II, leading to increased expression of downstream target genes such as glucose-6-phosphate isomerase. Collectively, we report a previously unrecognized role of the lncRNA NRCP in modulating cancer metabolism. As demonstrated, DOPC nanoparticle-incorporated siRNA-mediated silencing of this lncRNA in vivo provides therapeutic avenue toward modulating lncRNAs in cancer.

Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer.

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.

Abstract 4127: Bolus high-dose neoadjuvant sunitinib treatment overcomes potential for rebound growth during pre-surgical treatment breaks

Abstract Preclinical and clinical studies have suggested that stopping antiangiogenic therapy may lead to rapid revascularization and rebound tumor growth in certain instances. However, the implications for these findings remain unclear. It is possible that treatment rebounds may impact disease progression following neoadjuvant therapy, where drugs are administered for short periods and then halted prior to surgical removal of a primary tumor. Indeed, recent retrospective clinical studies have suggested that neoadjuvant antiangiogenic treatment discontinuation increases vascular proliferation and, in turn, might increase post-surgical disease progression and metastasis. However, this hypothesis has not been studied experimentally in clinically relevant mouse models of metastatic disease. We examined the effect of treatment discontinuation in the neoadjuvant setting using approved vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKIs) such as sunitinib. Our studies utilized models of spontaneous metastatic breast, melanoma, and kidney cancer following surgical removal of orthotopically grown primary tumors. Neoadjuvant sunitinib treatment periods were 14 days in duration (60 mg/kg) with treatment breaks of 0, 3, 7, and 11 days compared. Pre-surgical molecular and cellular biomarkers were investigated as potential surrogates for post-surgical metastatic disease progression. These included tumor and vascular cell Ki67 and CD31 expression, as well as circulating tumor cells (CTCs) and myeloid derived suppressor cells (MDSCs). Our results show that VEGF RTKI neoadjuvant treatment benefits observed pre-surgically (such as reduction in primary tumor growth) did not consistently correlate with post-surgical survival and reduced metastatic recurrence. The addition of treatment breaks prior to surgery had positive or negative impact on post-surgical survival depending on the tumor model used. Interestingly, high-dose bolus (3 day) neoadjuvant sunitinib (120 mg/kg) treatment consistently improved post-surgical benefits when compared to sustained treatments, despite gaps in therapy. Finally, our results show that tumoral CD31+/Ki67+ cells, CTCs, and MDSCs may serve as surrogate markers of metastatic potential and predict the impact of treatment cessation on disease recurrence. Citation Format: Michalis Mastri, Amanda Tracz, Li Shen, Roberto Pili, John M.L. Ebos. Bolus high-dose neoadjuvant sunitinib treatment overcomes potential for rebound growth during pre-surgical treatment breaks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4127. doi:10.1158/1538-7445.AM2015-4127