Orthotopic Breast Cancer Model Research Articles

Intratumorally delivered formulation, INT230-6, containing potent anticancer agents induces protective T cell immunity and memory

ABSTRACTThe benefits of anti-cancer agents extend beyond direct tumor killing. One aspect of cell death is the potential to release antigens that initiate adaptive immune responses. Here, a diffusion enhanced formulation, INT230-6, containing potent anti-cancer cytotoxic agents, was administered intratumorally into large (approx. 300mm3) subcutaneous murine Colon26 tumors. Treatment resulted in regression from baseline in 100% of the tumors and complete response in up to 90%. CD8+ or CD8+/CD4+ T cell double-depletion at treatment onset prevented complete responses, indicating a critical role of T cells in promoting complete tumor regression. Mice with complete response were protected from subcutaneous and intravenous re-challenge of Colon26 cells in a CD4+/CD8+ dependent manner. Thus, immunological T cell memory was induced by INT230-6. Colon26 tumors express the endogenous retroviral protein gp70 containing the CD8+ T-cell AH-1 epitope. AH-1-specific CD8+ T cells were detected in peripheral blood of tumor-bearing mice and their frequency increased 14 days after treatment onset. AH-1-specific CD8+ T cells were also significantly enriched in tumors of untreated mice. These cells had an activated phenotype and highly expressed Programmed cell-death protein-1 (PD-1) but did not lead to tumor regression. CD8+ T cell tumor infiltrate also increased 11 days after treatment. INT230-6 synergized with checkpoint blockade, inducing a complete remission of the primary tumors and shrinking of untreated contralateral tumors, which demonstrates not only a local but also systemic immunological effect of the combined therapy. Similar T-cell dependent inhibition of tumor growth was also found in an orthotopic 4T1 breast cancer model.

Abstract 2012: Interrogating the in vivo anti-metastatic action of tinzaparin and simvastatin in an orthotopic surgical resection mouse model of triple negative breast cancer

Abstract Background: One of the key events in the progression of cancer metastasis is the migration of tumor cells through the vascular endothelium. Tumors induce abnormal endothelial permeability to promote cancer cell invasion. Recently, inhibition of tumor-induced vascular permeability has been shown to attenuate metastasis in vivo (Tichet M et al. Nat Commun. 2015;6:6993). Low molecular weight heparins (LMWHs) inhibit metastasis in vivo (Klerk et al. J Clin Oncol. 2005;23(10):2130-5) but may cause bleeding complications when used at effective doses. Consequently, LMWHs are unlikely to be used for prevention of metastatic progression unless bleeding side-effects can be diminished. To address this issue, we have recently demonstrated that a LMWH/statin combination (tinzaparin and simvastatin) attenuates abnormal vascular permeability and reduces epithelial cell transmigration at concentrations unlikely to cause bleeding side-effects in vivo. (Kevane et al. Res Pract Thromb Haemost. 2017;1(1)). Here, we aim to interrogate the anti-metastatic effect of the LMWH/statin combination (tinzaparin and simvastatin) in an orthotopic surgical resection model of triple negative breast cancer implementing a LMWH/stain dose that does not increase bleeding risk. Methods: NOD/SCID mice were surgically implanted with 5x105 MDA-MB-231-LUC2 triple negative breast cancer cells in the right inguinal mammary fat pad. Tumors were allowed to grow until they reached 250mm3. Subsequently, primary tumors were surgically resected. 5 days post-resection, mice were randomized into 4 groups (group 1: 0.5% methylcellulose (vehicle), group 2: 150IU/kg TNZ, group 3: 3mg/kg SVS and group 4: TNZ (150IU/kg) and SVS (3mg/kg)) and treated daily for 4 weeks. Post-resection metastatic progression was monitored by weekly bioluminescence imaging (BLI) [IVIS Spectrum]. Animals that displayed metastatic progression or significant regrowth of primary tumour were euthanized. At time of death, apical lymph nodes and lungs were removed and fixed in 10% formalin for quantification of metastatic burden Results: Prior to study commencement it was determined that a combination of 150IU/kg TNZ and 3mg/kg SVS did not significantly increase bleeding time in a cohort of tumour naïve animals. Following primary tumor resection, BLI assessment of metastatic progression revealed that 150IU/kg TNZ, 3mg/kg SVS and combination therapy of 150IU/kg TNZ and 3mg/kg SVS significantly (P= 0.0373, P=0.0086 and P=0.0409 respectively N=11) increased the time to metastasis compared to the vehicle treated animals. Conclusion: For the first time we demonstrate that tinzaparin and simvastatin may be combined to significantly delay time to metastasis while diminishing the associated bleeding risk of LMWHs in a clinically relevant mouse model of aggressive triple negative breast cancer. Citation Format: Ian S. Miller, Liam P. Shiels, Grace Buckley, Kate Connor, Annette T. Byrne, Fionnuala Ní Ainle. Interrogating the in vivo anti-metastatic action of tinzaparin and simvastatin in an orthotopic surgical resection mouse model of triple negative breast cancer [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 2012.

Abstract 2015: Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis

Abstract Triple negative breast cancer (TNBC) is notoriously known for poor prognosis due to a lack of actionable molecular target(s). Remarkably, the majority of TNBC patients suffer the cancer relapse even they receive treatment given for early stage breast cancers. Up-to-date, lots of therapeutic agents have been developed and actionable molecular targets have been proposed. Nevertheless, the survival rate for metastatic invasive breast cancer is low because of poor prognosis. In this study, we determined whether different classes of chemo-agents (or compounds) interact with different cellular and molecular pathways they might share some common pathway(s) if their therapeutic outcome is similar. We first tested the effect of aminoguanidine (AG), APOE mimetics (COG), and nitroxyl (HNO) compounds on carcinogenesis and metastasis using an orthotopic breast cancer model. In vivo imaging analysis indicated that these compounds significantly reduced the tumor loading in the xenografts (MDA-MB-231-GFP). Moreover, compounds AG and COG are able to reduce brain metastasis significantly. RNA-Seq analysis identified only one gene which is commonly altered in all treatment groups. This gene encodes marginal zone B and B1 cell specific protein (MZB1). MZB1 was downregulated after treatment and its expression was inversely correlated with the efficacy of brain metastasis. In addition, pathway comparison analysis of RNA-Seq data revealed there are three top canonical pathways including (1) Role of NFAT in Regulation of the Immune Response, (2) PKC theta Signaling in T Lymphocytes, and (3) iCOS-iCOSL Signaling in T Helper Cells, downregulated in all treatment groups. In conclusion, our findings demonstrated three different classes of compounds can reduce not only tumor loading but metastasis. MZB1, identified from our transcriptome analysis, may serve as an actionable molecular target for TNBC. All-in-all, it provides new insights into the development of new therapeutic targets and biomarkers for TNBC. Citation Format: Yuk Sing Robert Cheng, Debashree Basudhar, Rdinour Lisa, Veena Somasundaram, David Wink. Downregulation of MZB1 by aminoguanidine, APOE mimetic and nitroxyl compounds associated with reduction in invasive breast tumor volume and metastasis [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 2015.

Abstract 1235: Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis

Abstract The majority of breast cancer-related mortality is due to metastasis. Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is overexpressed in breast cancer. Cytoplasmic HuR accumulation correlates with high-grade malignancy, poor distant disease-free survival and serves as a prognostic factor for poor clinical outcome in breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors and cytokines that implicate major tumor hallmarks including invasion and metastasis. Knocking out HuR by CRISPR/CAS9 technology inhibits cell invasion in breast cancer cells. Therefore, HuR is an emerging target for breast cancer therapy, especially the lethal metastatic breast cancer. RNA-binding proteins had previously been considered “undruggable” due to lack of a well-defined binding pocket for target RNAs. Using high throughput screening followed by structure-based rational design and lead optimization, we have identified small molecules that inhibit HuR-mRNA interaction at nM to sub-µM potency. Our lead compound, KH-3, potently inhibits breast cancer cell growth and decreases cell invasion in vitro similar to HuR knockout, as well as increasing the expression of epithelial marker E-cadherin. In the study of mechanism of action, a transcription factor, FOXQ1, which is recently revealed to implicate in breast cancer invasion and metastasis processes, is found for the first time to be a direct mRNA target of HuR and one of the top genes that are reduced by KH-3 treatment. Exogenous introduction of FOXQ1 can rescue cell invasive capability impaired by HuR knockout and abolish the effect of KH-3 on inhibiting cell invasion in breast cancer cells. Moreover, KH-3 disrupts HuR-FOXQ1 interaction in RNP-IP, RNA pull down and FOXQ1 3′-UTR luciferase reporter assays. In vivo efficacy studies show that KH-3 not only exhibits potent antitumor efficacy in an orthotopic xenograft model of breast cancer, but also efficiently inhibits lung metastasis and improves mouse survival in an experimental metastasis model. Our data provide a proof-of-principle that HuR inhibition by KH-3 may be developed as a promising molecular therapy for inhibiting progression and metastasis of breast cancer with high HuR. Citation Format: Xiaoqing Wu, Gulhumay Gardashova, Lan Lan, Shuang Han, Cuncong Zhong, Ragul Gowthaman, John Karanicolas, Dan A. Dixon, Danny R. Welch, Ling Li, Min Ji, Jeffrey Aubé, Liang Xu. Targeting RNA-binding protein HuR to inhibit human breast cancer invasion and metastasis [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 1235.

Abstract 2302: Natural killer cell cancer therapy in mouse model

Abstract Natural killer (NK) cells exert anti-tumor function through direct killing of tumor cells and indirect modulation of the function of other immune cells, such as T cells, dendritic cells and macrophages. However, the efficacy of adoptive NK cell transfer in cancer treatment has been limited, and only found in certain myeloid leukemia under allogeneic setting. The functional heterogeneity of NK cells and the immunosuppressive tumor microenvironment likely contribute to the limited efficacy. We establish a method to condition ex vivo expanded murine NK cells with anti-tumor activity. The conditioned NK cells are EOMES+CD127-DX5-CD49a+and CD11b+CD27+. Encountering with solid tumor cell lines in vitro enhances the production of IFN-γand triggers killing of the tumor cells by conditioned NK cells. In a syngeneic orthotopic EO771 breast cancer model, adoptive transfer of conditioned NK cells into tumor-bearing mice slows tumor progression and alters the composition and function of intra-tumor immune cells. To better imitate clinical condition, we transfer conditioned NK cells into tumor-resected mice, and found the therapy improved survival rate. We are investigating the anti-tumor mechanism of the conditioned NK cells. Citation Format: Shih-Wen Huang, Yein-Gei Lai, Jen-Qi Wu, Yae-Huei Liou, Nan-Shih Liao. Natural killer cell cancer therapy in mouse model [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 2302.

Abstract 3259: heterodimeric IL-15 monotherapy results in complete regression of EO771 murine breast tumors through cDC1-lymphocyte interactions and induction of antitumor immunity

Abstract Introduction: IL-15 is a cytokine important for the maintenance, proliferation and activation of lymphocytes, including CD8+ T and natural killer (NK) cells. The native form of IL-15 is a heterodimer either embedded in the plasma membrane or released in a soluble form. Several preclinical cancer models have supported the anti-tumor activity of heterodimeric IL-15, which is presently in clinical trials. We have produced heterodimeric IL-15 (hetIL-15) and also fusions to the Fc fragments (hetIL-15FC) of human, macaque or mouse antibodies. We assessed the effects of hetIL-15 monotherapy after systemic and locoregional administration in the proximity of EO771 orthotopically implanted breast tumors. Experimental procedures: We evaluated the therapeutic efficacy of hetIL-15 and hetIL-15FC immunotherapy in the murine EO771 triple negative breast cancer orthotopic model using both syngeneic C57BL/6 mice and RAG-1 immunodeficient mice. We monitored the effects of hetIL-15 treatment on Tumor Infiltrating Lymphocytes (TILs), lymphoid organs and blood by Flow cytometry and Immunohistochemisty (IHC). Using Seahorse technology, we also evaluated the metabolic profile of the CD8+ T cells, reflecting their activation status. Results: Monotherapy with hetIL-15FC peritumorally in the area of mammary pad resulted in complete tumor regression in half of the treated mice. hetIL-15 treatment resulted in an increased survival accompanied with decreased or completely eradicated metastatic disease, in C57BL/6 mice. Significant tumor delay was also observed after peritoneal injection of hetIL-15 in C57BL/6 as well as in RAG-1 mice with hetIL-15FC, but complete tumor regression was not achieved. hetIL-15FC peritumoral administration reshaped the tumor microenvironment and caused an increased infiltration of NK cells and CD8+ T cells with effector phenotype. Both cytotoxic T cells and NK were activated and proliferating, as shown by Flow, including Granzyme B and Ki67 staining. hetIL-15FC treatment resulted in increased tumor infiltration of both CD8+ and CD4+ T memory cells and provided strong protection to these mice against subsequent re-challenges with EO771 tumor. Furthermore, peritumoral hetIL-15FC administration resulted in an increase of intratumoral conventional Dendritic cells (cDC1), suggesting a mechanism for the intratumoral attraction of lymphocytes. Conclusion: Monotherapy with hetIL-15FC locoregional administration resulted in complete regression of EO771 primary breast cancer tumors, activating both cytotoxic arms of the immune system, and providing persisting protective immunity. Mechanism of function of hetIL-15 is through cDC1 and lymphocyte interactions that lead to the reciprocal attraction of cDC1 and activated cytotoxic lymphocytes into the tumors and the generation of long-term anti-tumor immunity. Citation Format: Dimitris Stellas, Sevasti Karaliota, Vasiliki Stravokefalou, Bethany A. Nagy, Barbara K. Felber, George N. Pavlakis. heterodimeric IL-15 monotherapy results in complete regression of EO771 murine breast tumors through cDC1-lymphocyte interactions and induction of antitumor immunity [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 3259.

Abstract 1963: Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression

Abstract One of the leading causes of non-responding tumors with existing immunotherapies is an immunosuppressive tumor micro-environment (TME). The suppressive nature of the TME is, in part, mediated by immunoregulatory cells (e.g. MDSC cells) and cytokines/chemokines. TGFB1 has recently been shown to be a key suppressive factor responsible for facilitating “cold” or immune-excluded tumors. In these tumors, T cell activity is restricted and by reducing TGFB1 levels we aim to support anti-tumor T cell activity. We evaluated if downregulation of TGFB1 with a self-delivering RNAi (sd-rxRNA) in MDSCs (in vitro) or TME (in vivo) promotes T cell activity and increase anti-tumor activity. sd-rxRNA is a chemically modified RNAi which incorporates drug like properties allowing for self-delivery of RNAi compounds to immune cells (including MDSC, DC, T and NK cells) in the absence of electroporation or formulation in a delivery vehicle. Tumor induced MDSCs were incubated with sd-rxRNA targeting TGFB1, its downstream target COX2 or in combination for 2 days. The cells were washed and added to a co-culture with OT-1 T cells and OVA expressing B16 melanoma. The anti-tumor activity of T-cells was monitored using a Real-Time Cell Activity assay (RTCA). TGFB1 and COX2 downregulated MDSCs had reduced immune-suppressive activity as shown by the enhanced B16-OVA tumor cell killing by CD8 OT-1 T cells in the RTCA assay while the combination of TGFB1 and COX2 MDSCs did not further improve the anti-tumor activity. To evaluate the effect of anti-TGFB1 sd-rxRNA in vivo, we first studied the tumor distribution and TGFB1 downregulation by intratumoral injection of sd-rxRNA to immunocompetent Balbc mice bearing 4T1-RL-GFP breast cancer in a fat pad model. DY547 labeled sd-rxRNA targeting PPIB or unlabeled sd-rxRNA targeting TGFB1 was injected into tumors. Two days later, tumors were resected, and fluorescence signal was analyzed by microscopy and TGFB1 downregulation was evaluated by RT-PCR. Efficient delivery of the DY547 labeled sd-rxRNA was observed in the vast majority of cells as shown by fluorescence microscopy. In addition, fluorescence signal could be detected in tumors up to 28 days after intratumoral injection, suggesting that sd-rxRNA was stable in tumors. Downregulation of TGFB1 in resected tumors, and an increased anti-tumor activity, was seen after three injections of sd-rxRNA. A follow up study was initiated investigating the efficacy of TGFB1 sd-rxRNA in combination with local tumor radiation on anti-tumor and metastatic activities in a 4T1 orthotopic breast cancer model. Preliminary data shows promising results. Our results suggest that sd-rxRNA compounds targeting TGFB1 can enhance the anti-tumor activity of T cells. Local injection of sd-rxRNA into tumors can effectively target TGFB1 in the TME resulting in less immunosuppression and increased anti-tumoral activity. Citation Format: Winnie F. Tam, Dingxue Yan, Melissa Maxwell, James Cardia, Gerrit Dispersyn. Feasibility and efficacy of using self-delivering RNAi against TGFB1 to reduce TME immunosuppression [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 1963.

Abstract 4553: Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow

Abstract Systemic immunosuppression in cancer is a mechanism of immune escape allowing for cancer progression to metastasis and remains a major pathophysiologic barrier to treatment including immunotherapies. The mechanism of this phenomenon remains relatively obscured. Although immune suppressive humoral factors secreted by primary tumors are thought to be a major cause of immune escape, a major question remains whether factors critical for cancer metastasis also dually regulate systemic immunosuppression to prepare peripheral sites for the eventual encounter with metastasizing cancer cells. To that end, we report here that the master cancer epithelial-mesenchymal transition (EMT) initiating factor Snail1, whose expression is restricted to the primary tumor where it promotes the generation of invasive breast tumor-initiating cells, yet also has a direct role in creating a pro-tumor systemic immune environment to promote metastasis. These dual roles for Snail1 in breast cancer progression has not been elucidated. Using both spontaneous genetic murine model of breast cancer and a syngeneic orthotopic breast cancer model we demonstrate that Snail1 is uniquely required for metastatic spread of breast tumor cells while also directly modulates the bone marrow niche, skewing it towards a suppressive immune phenotype with an upregulation of myeloid derived suppressor cells (MDSCs) and a decrease in antigen presenting cell (APC) populations. Importantly, deletion of Snail1 specifically in primary tumors severely abrogated metastasis and simultaneously restored bone marrow immune populations to levels in non-tumor bearing animals. In vitro co-culture assays demonstrated that humoral factors from Snail1-repleted cancer cells promoted MDSC formation and suppressed APCs when compared to Snail1-deficient cancer cells. Mechanistically, Snail1 modulates systemic immunosuppression specifically through 1) upregulating the expression of GM-CSF, CXCL2, and CCL2, factors important for MDSC induction and recruitment, within the primary tumor to saturate the periphery with these factors; and 2) generating metastatic cells that may suppress immune functions within the metastatic niche. Taken together, these data demonstrate Snail1’s central role in metastatic progression in breast cancer through its direct generation of metastatic cells and suppressing the systemic immune system to increase the chance that these metastatic cells may survive the periphery. Note: This abstract was not presented at the meeting. Citation Format: Mathew Sebastian, Dongjiang Chen, Son Le, Duy Nguyen, Changwang Deng, Dan Jin, Nagheme Thomas, David Tran. Snail1 in primary breast tumors remotely regulates a pro-tumor immune response in the bone marrow [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 4553.

Abstract 3931: The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment

Abstract Triple negative breast cancer (TNBC) recurrence rates remain high despite aggressive therapeutic intervention, including surgery, chemotherapy, and radiotherapy (RT). Recent studies suggest that circulating tumor cell re-seeding of primary tumors may facilitate recurrence rather than persistent tumor cells in the irradiated surgical bed. However, the role of the microenvironment in recurrence is not well understood. An emerging risk factor for breast cancer patients is lymphopenia or abnormally low systemic lymphocyte counts following therapy. In this study, we examined whether lymphopenia following therapy correlates to local recurrence. To investigate the relationship between prolonged low lymphocyte count and recurrence, we evaluated radiation effects on tumor and immune cell recruitment to normal tissues using an orthotopic breast cancer model of lymphopenia. We tested the hypothesis that local recurrence in some instances is due to the attraction of circulating tumor cells to irradiated tissues. Local cytokine secretion as well as protein expression levels were analyzed to evaluate how tumor-stromal interactions modulate tumor cell recruitment to sites of damage. Recurrence free survival at 5 years for TNBC patients with sustained low ALC 1-5 months after RT (n=37) was 62.5% compared with 97% for patients with normal ALC (n=46; p<0.0001). Ex vivo BLI analysis revealed that normal tissue irradiation attracted tumor cells to the mammary fat pad (MFP) and surrounding tissues in BALB/c mice with depleted CD8+ T cells. Macrophage infiltration was greatly enhanced in BALB/c mice with depleted CD8+ T cells (p<0.0001) and necessary for tumor cell recruitment. Luminex analysis of MFPs in mice lacking CD8+ T cells after RT showed enhanced cytokine secretion of factors that regulate the inflammatory microenvironment and influence tumor cell proliferation and invasion. RPPA analysis of irradiated MFPs in the absence of CD8+ T cells revealed factors that promote epithelial-mesenchymal transition. Taken together, these results demonstrate that the response of the tissue microenvironment to therapy is dependent on the immune milieu and may encourage local recurrence. Our study establishes the importance of considering tumor subtype and immune function when assessing failure and outcome risks in breast cancer. We show that normal tissue radiation response may play a role in modulating tumor and immune cell migration. These results suggest that the stroma facilitates local recurrence in breast cancer through influencing immune and tumor cell behavior following RT. Citation Format: Benjamin C. Hacker, Steven M. Alves, Dadi Jiang, Albert C. Koong, Amato J. Giaccia, Edward E. Graves, Marjan Rafat. The irradiated tissue microenvironment and its role in breast cancer recurrence: Enhanced macrophage infiltration promotes tumor cell recruitment [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 3931.

Durability of cell line xenograft resection models to interrogate tumor micro-environment targeting agents

Angiogenesis is a key tumor microenvironment (TME) event underpinning tumor growth and metastasis. Nevertheless, the relatively poor performance of anti-angiogenic therapies in clinical trials compared to pre-clinical studies implies that classical subcutaneous xenograft models have limited predictive potential in this setting. To address this issue, we established orthotopic surgical resection models of breast cancer, which replicate the phenotype of clinical post-resection micro-metastasis. To demonstrate the power and precision of these models, we recapitulated the BETH adjuvant trial (NCT00625898) where the addition of bevacizumab (BVZ) to chemotherapy plus trastuzumab (Trast) failed to provide additional benefit. SCID mice were orthotopically implanted with bioluminescent Her2+ MDA-MB-231 or HCC1954 cells and tumors resected c.5 weeks later. Following resection, mice were treated with 10 mg/kg Trast +5 mg/kg paclitaxel (PAC) IP once weekly for 6 cycles +/− weekly BVZ (5 mg/kg IP). Metastasis was monitored by imaging. Using these models our data confirms that the addition of the anti-angiogenic antibody BVZ to adjuvant Trast + chemotherapy provides no additional benefit compared with Trast + chemotherapy alone. Previous studies using non-resection subcutaneously engrafted xenografts failed to predict this outcome. Our results provide compelling evidence for the utility of cell line xenograft resection models to predict clinical outcome for TME targeting agents.

Tumor Site-Dependent Transport Properties Determine Nanotherapeutics Delivery and Its Efficacy

Insufficient delivery of systemically administered anticancer drugs to tumors can compromise therapeutic efficacy and develop drug delivery-based therapeutic resistance. Nanotherapeutics such as PEGylated liposomal doxorubicin (PLD) are designed to preferentially accumulate in tumors utilizing enhanced permeation and retention effect. However, their antitumor effects and resulting clinical outcomes are modest and heterogeneous among tumors. Here, we aimed to investigate whether the amount and efficacy of PLD delivered to tumors are tumor site dependent. We established orthotopic primary tumor or liver metastases models of murine breast cancer using 4 T1 cells. PLD showed significant therapeutic effects against tumors that grew in primary mammary sites but not in the liver. We found that differences in therapeutic efficacy were not because of the intrinsic biological resistance of cancer cells but rather were associated with tumor site-dependent differences in transport properties, such as the amount of PLD delivery, blood vessel function, relative vascular permeability, and mechanical pressure in tumors. Thus, transport properties in tumor is site dependent and can be used as phenotypic surrogate markers for tumor drug delivery and therapeutic efficacy.

Acid specific dark quencher QC1 pHLIP for multi-spectral optoacoustic diagnoses of breast cancer

Breast cancer is the most common type of malignant growth in women. Early detection of breast cancer, as well as the identification of possible metastatic spread poses a significant challenge because of the structural and genetic heterogeneity that occurs during the progression of the disease. Currently, mammographies, biopsies and MRI scans are the standard of care techniques used for breast cancer diagnosis, all of which have their individual shortfalls, especially when it comes to discriminating tumors and benign growths. With this in mind, we have developed a non-invasive optoacoustic imaging strategy that targets the acidic environment of breast cancer. A pH low insertion peptide (pHLIP) was conjugated to the dark quencher QC1, yielding a non-fluorescent sonophore with high extinction coefficient in the near infrared that increases signal as a function of increasing amounts of membrane insertion. In an orthotopic murine breast cancer model, pHLIP-targeted optoacoustic imaging allowed us to differentiate between healthy and breast cancer tissues with high signal/noise ratios. In vivo, the sonophore QC1-pHLIP could detect malignancies at higher contrast than its fluorescent analog ICG-pHLIP, which was developed for fluorescence-guided surgical applications. PHLIP-type optoacoustic imaging agents in clinical settings are attractive due to their ability to target breast cancer and a wide variety of other malignant growths for diagnostic purposes. Intuitively, these agents could also be used for visualization during surgery.

Pan-TAM Tyrosine Kinase Inhibitor BMS-777607 Enhances Anti-PD-1 mAb Efficacy in a Murine Model of Triple-Negative Breast Cancer.

Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PS→TAM receptor→PD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.

Optimization and Design of Magnetic Ferrite Nanoparticles with Uniform Tumor Distribution for Highly Sensitive MRI/MPI Performance and Improved Magnetic Hyperthermia Therapy.

Two major technical challenges of magnetic hyperthermia are quantitative assessment of agent distribution during and following administration and achieving uniform heating of the tumor at the desired temperature without damaging the surrounding tissues. In this study, we developed a multimodal MRI/MPI theranostic agent with active biological targeting for improved magnetic hyperthermia therapy (MHT). First, by systematically elucidating the magnetic nanoparticle magnetic characteristics and the magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) signal enhancement effects, which are based on the magnetic anisotropy, size, and type of nanoparticles, we found that 18 nm iron oxide NPs (IOs) could be used as superior nanocrystallines for high performance of MRI/MPI contrast agents in vitro. To improve the delivery uniformity, we then targeted tumors with the 18 nm IOs using a tumor targeting peptide, CREKA. Both MRI and MPI signals showed that the targeting agent improves the intratumoral delivery uniformity of nanoparticles in a 4T1 orthotopic mouse breast cancer model. Lastly, the in vivo antitumor MHT effect was evaluated, and the data showed that the improved targeting and delivery uniformity enables more effective magnetic hyperthermia cancer ablation than otherwise identical, nontargeting IOs. This preclinical study of image-guided MHT using cancer-targeting IOs and a novel MPI system paves the way for new MHT strategies.

Targeting DAMP-induced inflammation to prevent breast cancer metastasis and improve anti-tumor immunity

Abstract Biomarkers associated with systemic inflammation correlate with poor prognosis in cancer patients. Circulating cell-free DNA (cfDNA) is released passively and actively by both tumor cells and host cells and cfDNA levels in blood correlate with tumor burden in many tumor types. Notably, studies in breast cancer have shown that inhibition of cfDNA has a significant effect in reducing invasion and migration in vitro and metastatic disease in vivo. We have previously demonstrated that nucleic acid binding polymers (NABPs) bind and inhibit the proinflammatory effects of cfDNA in a murine model of pancreatic cancer. We observed that levels of cfDNA in blood increase with tumor burden in an orthotopic immunocompetent murine model of breast cancer. Moreover, treatment of mice with the NABP, PAMAM-G3, following surgical removal of primary tumor results in decreased lung metastasis. This decrease in lung metastasis is associated with reduction in the levels of cfDNA in blood. In addition, treatment of mice with NABPs in an intravenous experimental metastasis breast cancer model results in reduced seeding of tumor cells in lungs and lung metastasis. Analysis of immune infiltrates in lungs of PAMAM-G3- and control-treated mice demonstrates NABP-mediated transition of immune cell composition in lungs from innate to adaptive immune cells. Collectively, these data suggest that NABPs can be used to target cfDNA and inhibit DAMPs associated pathological inflammation. The use of NABPs is an innovative approach to treat cancer progression and metastasis, particularly in a clinical setting following standard of care surgical tumor resection. This work is funded by the Department of Defense Breast Cancer Research Program award (PI, Smita Nair).

Tracking the multiple-step formation of an iron(III) complex and its application in photodynamic therapy for breast cancer

A tandem reaction of pyridin-2-ylmethanamine (L1′) with 8-hydroxyquinoline-2-carbaldehyde (HL1) assisted by FeCl3 was observed to give the new nitrogen heterocycle HL3 (HL3=2-(imidazo[1,5-a]pyridin-3-yl)quinolin-8-ol) as its Fe(III) complex, [Fe(L3)Cl2] (Fel). Electrospray ionization mass spectrometry (ESI-MS) reveals its formation involves three steps: (1) coordination of both HL1 and Ll′ to Fe, (2) aldehyde-amine coupling, and (3) ring closure. The results of electronic absorption spectroscopy, cyclic voltammetry, and density functional theory (DFT) calculations show the proximity of the optical transition energy to that of the excitation of 3O2 to 1O2, which prompted us to explore its application as a photosensitizer for photodynamic therapy (PDT). Photo-toxicity studies show that Fel exhibits the highest anti-proliferation efficiency in human breast cancer MDA-MB-231 cells under light irradiation. Moreover, studies with orthotopic models of breast cancer further expounded the anti-tumor activity of Fe1 with no significant toxicity to other organs and low retention in the body.

Expanding therapeutic utility of carfilzomib for breast cancer therapy by novel albumin-coated nanocrystal formulation

Carfilzomib (CFZ) is the second-in-class proteasome inhibitor with much improved efficacy and safety profiles over bortezomib in multiple myeloma patients. In expanding the utility of CFZ to solid cancer therapy, the poor aqueous solubility and in vivo instability of CFZ are considered major drawbacks. We investigated whether a nanocrystal (NC) formulation can address these issues and enhance anticancer efficacy of CFZ against breast cancer. The surface of NC was coated with albumin in order to enhance the formulation stability and drug delivery to tumors via interactions with albumin-binding proteins located in and near cancer cells. The novel albumin-coated NC formulation of CFZ (CFZ-alb NC) displayed improved metabolic stability and enhanced cellular interactions, uptake and cytotoxic effects in breast cancer cells in vitro. Consistently, CFZ-alb NC showed greater anticancer efficacy in a murine 4T1 orthotopic breast cancer model than the currently used cyclodextrin-based formulation. Overall, our results demonstrate the potential of CFZ-alb NC as a viable formulation for breast cancer therapy.

Dual functional immunostimulatory polymeric prodrug carrier with pendent indoximod for enhanced cancer immunochemotherapy

Immunotherapy based on checkpoint blockade has been regarded as one of the most promising approaches towards many types of cancers. However, low response rate hinders its application due to insufficient tumor immunogenicity and immunosuppressive tumor microenvironment. To achieve an overall enhanced therapeutic outcome, we developed a dual-functional immuno-stimulatory polymeric prodrug carrier modified with pendent indoximod, an indoleamine 2,3-dioxygenase (IDO) inhibitor that can be used to reverse immune suppression, for co-delivery of Doxorubicin (Dox), a hydrophobic anticancer agent that can promote immunogenic cell death (ICD) and elicit antitumor immunity. The resulted carrier denoted as POEG-b-PVBIND, consisting of poly (oligo (ethylene glycol) methacrylate) (POEG) hydrophilic blocks and indoximod conjugated hydrophobic blocks, is rationally designed to improve immunotherapy by synergistically modulating the tumor microenvironment (TME). Our data showed that Dox-triggered ICD promoted intra-tumoral infiltration of CD8+ T cells and IFN-γ-production by CD8+ T cells. Meanwhile, cleaved indoximod significantly increased CD8+ T cell infiltration while reducing the immunosuppressive T regulatory cells (Tregs). More importantly, Dox/POEG-b-PVBIND micelles led to significantly improved tumor regression in an orthotopic murine breast cancer model compared to both Dox-loaded POEG-b-PVB micelles (a control inert carrier) and POEG-b-PVBIND micelles alone, confirming combination effect of indoximod and Dox in improving the overall antitumor activity. Statement of SignificanceIndoleamine 2,3-dioxygenase (IDO) is an enzyme that can induce immune suppressive microenvironment in tumors. As a well-studied IDO inhibitor, indoximod (IND) represents a promising agent for cancer immunotherapy and could be particularly useful in combination with other chemotherapeutic agents. However, three major problems hinder its application: (1) IND is barely soluble in water; (2) IND delivery efficiency is limited (3) simultaneous delivery of two agents into tumor site is still challenging. Currently, most reports largely focus on improving the pharmacokinetic profile of IND alone via different formulations such as IND prodrug and IND nanocrystal. However, there is limited information about IND based co-delivery systems, especially for delivering hydrophobic chemotherapeutic agents. Here, we developed a new dual-functional polymeric prodrug carrier modified with a number of pendent IND units (denoted as POEG-b-PVBIND). POEG-b-PVBIND shows immunostimulatory and antitumor activities by itself. More importantly, POEG-b-PVBIND polymer is able to self-assemble into nano-sized micelles that are highly effective in formulating and codelivering other hydrophobic agents including doxorubicin (Dox), sunitinib (Sun), and daunorubicin (Dau), which can elicit antitumor immunity via promoting immunogenic cell death (ICD). We have shown that our new combination therapy led to a significantly improved antitumor activity in an aggressive murine breast cancer model (4T1.2).

ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis

Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.

Stromal integrin \u03b111-deficiency reduces interstitial fluid pressure and perturbs collagen structure in triple-negative breast xenograft tumors

BackgroundCancer progression is influenced by a pro-tumorigenic microenvironment. The aberrant tumor stroma with increased collagen deposition, contractile fibroblasts and dysfunctional vessels has a major impact on the interstitial fluid pressure (PIF) in most solid tumors. An increased tumor PIF is a barrier to the transport of interstitial fluid into and within the tumor. Therefore, understanding the mechanisms that regulate pressure homeostasis can lead to new insight into breast tumor progression, invasion and response to therapy. The collagen binding integrin α11β1 is upregulated during myofibroblast differentiation and expressed on fibroblasts in the tumor stroma. As a collagen organizer and a probable link between contractile fibroblasts and the complex collagen network in tumors, integrin α11β1 could be a potential regulator of tumor PIF.MethodsWe investigated the effect of stromal integrin α11-deficiency on pressure homeostasis, collagen organization and tumor growth using orthotopic and ectopic triple-negative breast cancer xenografts (MDA-MB-231 and MDA-MB-468) in wild type and integrin α11-deficient mice. PIF was measured by the wick-in-needle technique, collagen by Picrosirius Red staining and electron microscopy, and uptake of radioactively labeled 5FU by microdialysis. Further, PIF in heterospheroids composed of MDA-MB-231 cells and wild type or integrin α11-deficient fibroblasts was measured by micropuncture.ResultsStromal integrin α11-deficiency decreased PIF in both the orthotopic breast cancer models. A concomitant perturbed collagen structure was seen, with fewer aligned and thinner fibrils. Integrin α11-deficiency also impeded MDA-MB-231 breast tumor growth, but no effect was observed on drug uptake. No effects were seen in the ectopic model. By investigating the isolated effect of integrin α11-positive fibroblasts on MDA-MB-231 cells in vitro, we provide evidence that PIF regulation was mediated by integrin α11-positive fibroblasts.ConclusionWe hereby show the importance of integrin α11β1 in pressure homeostasis in triple-negative breast tumors, indicating a new role for integrin α11β1 in the tumor microenvironment. Our data suggest that integrin α11β1 has a pro-tumorigenic effect on triple-negative breast cancer growth in vivo. The significance of the local microenvironment is shown by the different effects of integrin α11β1 in the orthotopic and ectopic models, underlining the importance of choosing an appropriate preclinical model.