Programable Albumin-Hitchhiking Nanobodies Enhance the Delivery of STING Agonists to Potentiate Cancer Immunotherapy.

Stimulator of interferon genes (STING) is a promising target for potentiating antitumor immunity, but multiple pharmacological barriers limit the clinical utility, efficacy, and/or safety of STING agonists. Here we describe a modular platform for systemic administration of STING agonists based on nanobodies engineered for in situ hitchhiking of agonist cargo on serum albumin. Using site-selective bioconjugation chemistries to produce molecularly defined products, we found that covalent conjugation of a STING agonist to anti-albumin nanobodies improved pharmacokinetics and increased cargo accumulation in tumor tissue, stimulating innate immune programs that increased the infiltration of activated natural killer cells and T cells, which potently inhibited tumor growth in multiple mouse tumor models. We also demonstrated the programmability of the platform through the recombinant integration of a second nanobody domain that targeted programmed cell death ligand-1 (PD-L1), which further increased cargo delivery to tumor sites while also blocking immunosuppressive PD-1/PD-L1 interactions. This bivalent nanobody carrier for covalently conjugated STING agonists stimulated robust antigen-specific T cell responses and long-lasting immunological memory, conferred enhanced therapeutic efficacy, and was effective as a neoadjuvant treatment for improving responses to adoptive T cell transfer therapy. Albumin-hitchhiking nanobodies thus offer an enabling, multimodal, and programmable platform for systemic delivery of STING agonists with potential to augment responses to multiple immunotherapeutic modalities.

Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic Modulation.

Interleukin-4 (IL4) is a Th2 cytokine that can signal through two different receptors, one of which-the type II receptor-is overexpressed by various cancer cells. Previously, we have shown that type II IL4 receptor signaling increases proliferation and metastasis in mouse models of breast cancer, as well as increasing glucose and glutamine metabolism. Here, we expand on those findings to determine mechanistically how IL4 signaling links glucose metabolism and histone acetylation to drive proliferation in the context of triple-negative breast cancer (TNBC). We used a combination of cellular, biochemical, and genomics approaches to interrogate TNBC cell lines, which represent a cancer type where high expression of the type II IL4 receptor is linked to reduced survival. Our results indicate that type II IL4 receptor activation leads to increased glucose uptake, Akt and ACLY activation, and histone acetylation in TNBC cell lines. Inhibition of glucose uptake through the deletion of Glut1 ablates IL4-induced proliferation. Additionally, pharmacological inhibition of histone acetyltransferase P300 attenuates IL4-mediated gene expression and proliferation in vitro. Our work elucidates a role for type II IL4 receptor signaling in promoting TNBC progression, and highlights type II IL4 signaling, as well as histone acetylation, as possible targets for therapy.

Tumor therapy by targeting extracellular hydroxyapatite using novel drugs: A paradigm shift.

BACKGROUND: It has been shown that tumor microenvironment (TME) hydroxyapatite (HAP) is typically associated with many malignancies and plays a role in tumor progression and growth. Additionally, acidosis in the TME has been reported to play a key role in selecting for a more aggressive tumor phenotype, drug resistance and desensitization to immunotherapy for many types of cancers. TME-HAP is an attractive target for tumor detection and treatment development since HAP is generally absent from normal soft tissue. We provide strong evidence that dissolution of hydroxyapatite (HAP) within the tumor microenvironment (TME-HAP) using a novel therapeutic can be used to kill cancer cells both in vitro and in vivo with minimal adverse effects. METHODS: We developed an injectable cation exchange nano particulate sulfonated polystyrene solution (NSPS) that we engineered to dissolve TME-HAP, inducing localized acute alkalosis and inhibition of tumor growth and glucose metabolism. This was evaluated in cell culture using 4T1, MDA-MB-231 triple negative breast cancer cells, MCF10 normal breast cells, and H292 lung cancer cells, and in vivo using orthotopic mouse models of cancer that contained detectable microenvironment HAP including breast (MMTV-Neu, 4T1, and MDA-MB-231), prostate (PC3) and colon (HCA7) cancer using 18 F-NaF for HAP and 18 F-FDG for glucose metabolism with PET imaging. On the other hand, H292 lung tumor cells that lacked detectable microenvironment HAP and MCF10a normal breast cells that do not produce HAP served as negative controls. Tumor microenvironment pH levels following injection of NSPS were evaluated via Chemical Exchange Saturation (CEST) MRI and via ex vivo methods. RESULTS: Within 24 h of adding the small concentration of 1X of NSPS (~7 µM), we observed significant tumor cell death (~ 10%, p < 0.05) in 4T1 and MDA-MB-231 cell cultures that contain HAP but ⟨2% in H292 and MCF10a cells that lack detectable HAP and in controls. Using CEST MRI, we found extracellular pH (pHe) in the 4T1 breast tumors, located in the mammary fat pad, to increase by nearly 10% from baseline before gradually receding back to baseline during the first hour post NSPS administration. in the tumors that contained TME-HAP in mouse models, MMTV-Neu, 4T1, and MDA-MB-231, PC3, and HCA7, there was a significant reduction (p<0.05) in 18 F-Na Fuptake post NSPS treatment as expected; 18 F- uptake in the tumor = 3.8 ± 0.5 %ID/g (percent of the injected dose per gram) at baseline compared to 1.8 ±0.5 %ID/g following one-time treatment with 100 mg/kg NSPS. Of similar importance, is that 18 F-FDG uptake in the tumors was reduced by more than 75% compared to baseline within 24 h of treatment with one-time NSPS which persisted for at least one week. Additionally, tumor growth was significantly slower (p < 0.05) in the mice treated with one-time NSPS. Toxicity showed no evidence of any adverse effects, a finding attributed to the absence of HAP in normal soft tissue and to our therapeutic NSPS having limited penetration to access HAP within skeletal bone. CONCLUSION: Dissolution of TME-HAP using our novel NSPS has the potential to provide a new treatment paradigm to enhance the management of cancer patients with poor prognosis.

Asthma is associated with a lower incidence of metastatic colorectal cancer in a US patient cohort.

In previous pre-clinical studies, we examined the contribution of interleukin 4 receptor (IL4R) signaling in the progression and metastasis of colorectal cancer (CRC). Aberrant activation of this receptor can result in atopic diseases such as asthma. We hypothesized that further evidence for the contribution of excessive IL4R being associated with CRC progression could be seen in medical records, and specifically that chronic asthma patients were more likely to be diagnosed with metastatic CRC. To test this hypothesis, we took advantage of the Synthetic Derivative, a resource developed at Vanderbilt University Medical Center that hosts de-identified data taken from the electronic medical record. We developed search protocols that produced retrospective cohorts of invasive CRC patients and cancer-free equivalents. In comparing 787 metastatic CRC patients to 238 non-metastatic patients, we actually found significantly fewer asthmatics went on to develop metastatic CRC (P=0.0381). By comparing these groups together against 1197 cancer-free patients, even fewer asthmatic patients would develop invasive CRC (P<0.0001). While these results are clearly in opposition to our original hypothesis, they still support a link between chronic asthma and metastatic CRC development. One intriguing possibility, that will be examined in the future, is whether treatment for chronic asthma may be responsible for the reduction in metastatic cancer.

Sex Hormones in Breast Cancer Immunity.

Sex hormones, such as estrogens and androgens, regulate genomic and cellular processes that contribute to sex-specific disparities in the pathophysiology of various cancers. Sex hormones can modulate the immune signals and activities of tumor cells and tumor-associated leukocytes to support or suppress cancer progression. Therefore, hormonal differences between males and females play a crucial role in cancer immunity and in the response to therapies that exploit the intrinsic immune system to eliminate malignant cells. In this review, we summarize the impact of sex hormones in the breast cancer microenvironment, with a focus on how the hormonal environment affects tumor immunity. We also discuss the potential benefits of endocrine therapy used in combination with immunotherapy to strengthen the antitumor immune response.

The therapeutic potential of TREM2 in cancer.

Cancer continues to be a substantial health concern and a leading cause of death in the United States and around the world. Therefore, it is important to continue to explore the potential of novel therapeutic targets and combinatorial therapies. Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor of the immunoglobulin superfamily that associates with DNAX activation protein (DAP) 12 and DAP10 to propagate signals within the cell. TREM2 has primarily been recognized for its expression on cells in the monocyte-macrophage lineage, with the majority of work focusing on microglial function in Alzheimer's Disease. However, expansion of TREM2 research into the field of cancer has revealed that epithelial tumor cells as well as intratumoral macrophages and myeloid regulatory cells also express TREM2. In this review, we discuss evidence that TREM2 contributes to tumor suppressing or oncogenic activity when expressed by epithelial tumor cells. In addition, we discuss the immunosuppressive role of TREM2-expressing intratumoral macrophages, and the therapeutic potential of targeting TREM2 in combination with immune checkpoint therapy. Overall, the literature reveals TREM2 could be considered a novel therapeutic target for certain types of cancer.

Glycosylation as a regulator of site-specific metastasis.

Metastasis is considered to be responsible for 90% of cancer-related deaths. Although it is clinically evident that metastatic patterns vary by primary tumor type, the molecular mechanisms underlying the site-specific nature of metastasis are an area of active investigation. One mechanism that has emerged as an important player in this process is glycosylation, or the addition of sugar moieties onto protein and lipid substrates. Glycosylation is the most common post-translational modification, occurring on more than 50% of translated proteins. Many of those proteins are either secreted or expressed on the cell membrane, thereby making glycosylation an important mediator of cell-cell interactions, including tumor-microenvironment interactions. It has been recently discovered that alteration of glycosylation patterns influences cancer metastasis, both globally and in a site-specific manner. This review will summarize the current knowledge regarding the role of glycosylation in the tropism of cancer cells for several common metastatic sites, including the bone, lung, brain, and lymph nodes.

Measurement of Metabolites from Migrating Cells.

Metastasis is a multistep process that involves responses to extrinsic and intrinsic signals at every step. It is thus only truly appreciated in the context of a whole organism. Nevertheless, in vitro studies can be used to facilitate understanding of the possible factors contributing to any phenotype that is associated with metastatic competence. The use of migration assays-where monolayers of cells migrate to cover gaps or "wounds"-has been described for decades to identify signaling pathways that regulate motile competence and to screen for ways of interfering with this ability. Here we depict the combination of such an assay with assessment of indicators of carbon metabolism using commercially available assays. This enables identification of changes in cellular metabolism associated with actively migrating cells.

Development of a novel murine model of lymphatic metastasis.

Current laboratory models of lymphatic metastasis generally require either genetically modified animals or are technically challenging. Herein, we have developed a robust protocol for the induction of intralymphatic metastasis in wild-type mice with reproducible outcomes. To determine an optimal injection quantity and timeline for tumorigenesis, C57Bl/6 mice were injected directly into the mesenteric lymph duct (MLD) with varying numbers of syngeneic murine colon cancer cells (MC38) or gastric cancer cells (YTN16) expressing GFP/luciferase and monitored over 2-4 weeks. Tumor growth was tracked via whole-animal in vivo bioluminescence imaging (IVIS). Our data indicate that the injection of tumor cells into the MLD is a viable model for lymphatic metastasis as necropsies revealed large tumor burdens and metastasis in regional lymph nodes. This protocol enables a closer study of the role of lymphatics in cancer metastasis and opens a window for the development of novel approaches for treatment of metastatic diseases.

The importance of developing therapies targeting the biological spectrum of metastatic disease.

Great progress has been made in cancer therapeutics. However, metastasis remains the predominant cause of death from cancer. Importantly, metastasis can manifest many years after initial treatment of the primary cancer. This is because cancer cells can remain dormant before forming symptomatic metastasis. An important question is whether metastasis research should focus on the early treatment of metastases, before they are clinically evident ("overt"), or on developing treatments to stop overt metastasis (stage IV cancer). In this commentary we want to clarify why it is important that all avenues of treatment for stage IV patients are developed. Indeed, future treatments are expected to go beyond the mere shrinkage of overt metastases and will include strategies that prevent disseminated tumor cells from emerging from dormancy.

Non-canonical roles for metabolic enzymes and intermediates in malignant progression and metastasis.

Metabolic alterations are established as a hallmark of cancer. Such hallmark changes in cancer metabolism are characterized by reprogramming of energy-producing pathways and increases in the generation of biosynthetic intermediates to meet the needs of rapidly proliferating tumor cells. Various metabolic phenotypes such as aerobic glycolysis, increased glutamine consumption, and lipolysis have also been associated with the process of metastasis. However, in addition to the energy and biosynthetic alterations, a number of secondary functions of enzymes and metabolites are emerging that specifically contribute to metastasis. Here, we describe atypical intracellular roles of metabolic enzymes, extracellular functions of metabolic enzymes, roles of metabolites as signaling molecules, and epigenetic regulation mediated by altered metabolism, all of which can affect metastatic progression. We highlight how some of these mechanisms are already being exploited for therapeutic purposes, and discuss how others show similar potential.

Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic ß-catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling is unknown. Here, we confirm that BVES loss increases ß-catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modeling using a Wnt-driven genetic model and a chemically induced model of colorectal carcinogenesis demonstrate that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.

Matrix metalloproteinases as regulators of inflammatory processes.

Inflammation is a central mechanism for dealing with insults to tissue, either from pathogenic invaders or by other damage-inducing means, such that the threat is removed, the tissue is healed and there is a return to homeostasis. It is a multi-step process with manifold methods of regulation built in. Proteolysis is one such regulatory method and members of the matrix metalloproteinase (MMP) family of proteinases have been shown to influence inflammation in myriad of ways. It is becoming more and more clear that no single MMP can be unequivocally labeled as 'good' or 'bad' when considering inflammation in general - the net result of proteolytic activity is dependent on context. Here we provide examples from recent literature, with a focus on in vivo studies, to highlight this concept. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Meeting report: Metastasis Research Society-Chinese Tumor Metastasis Society joint conference on metastasis.

During September 16th-20th 2016, metastasis experts from around the world convened for the 16th Biennial Congress of the Metastasis Research Society and 12th National Congress of the Chinese Tumor Metastasis Society in Chengdu, China to share most current data covering basic, translational, and clinical metastasis research. Presentations of the more than 40 invited speakers of the main congress and presentations from the associated Young Investigator Satellite Meeting are summarized in this report by session topic. The congress program also included three concurrent short talk sessions, an advocacy forum with Chinese and American metastatic patient advocates, a 'Meet the Professors Roundtable' session for young investigators, and a 'Meet the Editors' session with editors from Cancer Cell and Nature Cell Biology. The goal of integrating expertise and exchanging the latest findings, ideas, and practices in cancer metastasis research was achieved magnificently, thanks to the excellent contributions of many leaders in the field.

Perspective on the interpretation of research and translation to clinical care with therapy-associated metastatic breast cancer progression as an example.

This commentary was written as a collaboration between the Board of the Metastasis Research Society and two patients with metastatic breast cancer. It was conceived in response to how preclinical scientific research is sometimes presented to non-scientists in a way that can cause stress and confusion. Translation of preclinical findings to the clinic requires overcoming multiple barriers. This is irrespective of whether the findings relate to exciting responses to new therapies or problematic effects of currently used therapies. It is important that these barriers are understood and acknowledged when research findings are summarized for mainstream reporting. To minimize confusion, patients should continue to rely on their oncology care team to help them interpret whether research findings presented in mainstream media have relevance for their individual care. Researchers, both bench and clinical, should work together where possible to increase options for patients with metastatic disease, which is still in desperate need of effective therapeutic approaches.

Targeting IL4/IL4R for the treatment of epithelial cancer metastasis.

While progress has been made in treating primary epithelial tumors, metastatic tumors remain largely incurable and still account for 85-90 % of all cancer-related deaths. Interleukin-4 (IL4), a Th2 cytokine, and the IL4/IL4 receptor (IL4R) interaction have well defined roles in the immune system. Yet, IL4 receptors are over-expressed by many epithelial cancers and could be a promising target for metastatic tumor therapy. The IL4/IL4R signaling axis is a strong promoter of pro-metastatic phenotypes in epithelial cancer cells including enhanced migration, invasion, survival, and proliferation. The promotion of breast cancer growth specifically is also supported in part by IL4-induced glutamine metabolism, and we have shown that IL4 is also capable of inducing glucose metabolism in breast cancer cells. Importantly, there are several types of FDA approved medications for use in asthma patients that inhibit the IL4/IL4R signaling axis. However, these approved medications inhibit both the type I IL4 receptor found on immune cells, and the type II IL4 receptor that is predominantly expressed by some non-hematopoietic cells including epithelial cancer cells. This article reviews existing therapies targeting IL4, IL4R, or IL4/IL4R signaling, and recent findings guiding the creation of novel therapies that specifically inhibit the type II IL4R, while taking into consideration effects on immune cells within the tumor microenvironment. Some of these therapies are currently in clinical trials for cancer patients, and may be exploitable for the treatment of metastatic disease.

Selenoprotein P influences colitis-induced tumorigenesis by mediating stemness and oxidative damage.

Patients with inflammatory bowel disease are at increased risk for colon cancer due to augmented oxidative stress. These patients also have compromised antioxidant defenses as the result of nutritional deficiencies. The micronutrient selenium is essential for selenoprotein production and is transported from the liver to target tissues via selenoprotein P (SEPP1). Target tissues also produce SEPP1, which is thought to possess an endogenous antioxidant function. Here, we have shown that mice with Sepp1 haploinsufficiency or mutations that disrupt either the selenium transport or the enzymatic domain of SEPP1 exhibit increased colitis-associated carcinogenesis as the result of increased genomic instability and promotion of a protumorigenic microenvironment. Reduced SEPP1 function markedly increased M2-polarized macrophages, indicating a role for SEPP1 in macrophage polarization and immune function. Furthermore, compared with partial loss, complete loss of SEPP1 substantially reduced tumor burden, in part due to increased apoptosis. Using intestinal organoid cultures, we found that, compared with those from WT animals, Sepp1-null cultures display increased stem cell characteristics that are coupled with increased ROS production, DNA damage, proliferation, decreased cell survival, and modulation of WNT signaling in response to H2O2-mediated oxidative stress. Together, these data demonstrate that SEPP1 influences inflammatory tumorigenesis by affecting genomic stability, the inflammatory microenvironment, and epithelial stem cell functions.

Host and tumor derived MMP13 regulate extravasation and establishment of colorectal metastases in the liver.

BACKGROUND: Non alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the United States and worldwide. Our studies have previously shown an increase in metastatic burden in steatotic vs. normal livers using a mouse model of diet induced steatosis. In the present study we aim to identify and evaluate the molecular factors responsible for this increase in tumor burden. METHODS: We assessed changes in expression of a panel of matrix metalloproteinases (MMPs) using qRT-PCR between normal and steatotic livers and validated them with western blot analysis of protein levels. To evaluate the role of MMP13 on tumor development, we utilized a splenic injection model of liver metastasis in Wildtype and Mmp13 deficient mice, using either parental or stable Mmp13 knockdown cell lines. Further, to evaluate changes in the ability of tumor cells to extravasate we utilized whole organ confocal microscopy to identify individual tumor cells relative to the vasculature. MTT, migration and invasion assays were performed to evaluate the role of tumor derived MMP13 on hallmarks of cancer in vitro. RESULTS: We found that MMP13 was significantly upregulated in the steatotic liver both in mice as well as human patients with NAFLD. We showed a decrease in metastatic tumor burden in Mmp13-/- mice compared to wildtype mice, explained in part by a reduction in the number of tumor cells extravasating from the hepatic vasculature in the Mmp13-/- mice compared to wildtype mice. Additionally, loss of tumor derived MMP13 through stable knockdown in tumor cell lines lead to decreased migratory and invasive properties in vitro and metastatic burden in vivo. CONCLUSIONS: This study demonstrates that stromal as well as tumor derived MMP13 contribute to tumor cell extravasation and establishment of metastases in the liver microenvironment.

IL4 receptor α mediates enhanced glucose and glutamine metabolism to support breast cancer growth.

The type II interleukin-4 receptor (IL4R) is expressed in human breast cancer, and in murine models thereof. It is activated by interleukin-4 (IL4), a cytokine produced predominantly by immune cells. Previously, we showed that expression of IL4Rα, a signaling component of IL4R, mediates enhanced metastatic growth through promotion of tumor cell survival and proliferation. In lymphocytes, these processes are supported by increased glucose and glutamine metabolism, and B lymphocyte survival is dependent upon IL4/IL4R-induced glucose metabolism. However, it is unknown whether IL4R-mediated metabolic reprogramming could support tumor growth. Here, we show that IL4Rα expression increases proliferation thus enhancing primary mammary tumor growth. In vitro, IL4-enhanced glucose consumption and lactate production in 4T1 cells was mediated by IL4Rα. Expression of the glucose transporter GLUT1 increased in response to IL4 in vitro, and enhanced GLUT1 expression was associated with the presence of IL4Rα in 4T1 mammary tumors in vivo. Although IL4 treatment did not induce changes in glucose metabolism in MDA-MB-231 human breast cancer cells, it increased expression of the main glutamine transporter, ASCT2, and enhanced glutamine consumption in both MDA-MB-231 and 4T1 cells. Pharmacologic inhibition of glutamine metabolism with compound 968 blocked IL4/IL4Rα-increased cell number in both cell lines. Our results demonstrate that IL4R mediates enhanced glucose and glutamine metabolism in 4T1 cancer cells, and that IL4-induced growth is supported by IL4/IL4R-enhanced glutamine metabolism in both human and murine mammary cancer cells. This highlights IL4Rα as a possible target for effective breast cancer therapy.