Development of an in vivo system to model breast cancer metastatic organotropism and evaluate treatment response using the chick embryo.

Metastatic lesions produced through the process of systemic tumor cell dissemination and growth at distant sites are challenging to treat and the primary cause of patient mortality. Developing in vivo models of metastasis with utility in evaluating molecular targets and therapeutics in a timely manner would expedite the path to therapeutic discovery. Here, we evaluated breast cancer metastasis and metastatic organotropism using the chick embryo. We developed a method to evaluate metastasis using the MDA231 cell line. Then, using cell lines with demonstrated tropism for the bone, brain, and lung, we evaluated organotropism. Rapid and robust organ-specific metastasis was modeled in the chick embryo and, importantly, recapitulated metastatic organotropism congruent to what has been demonstrated in mice. Treatment response in the metastatic setting was also evaluated and quantified. This work establishes the chick embryo as a model for studies aimed at understanding organotropism and therapeutic response in the metastatic setting.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity.

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

Longitudinal bioluminescence imaging to monitor breast tumor growth and treatment response using the chick chorioallantoic membrane model.

The development of successful treatment regimens for breast cancer requires strong pre-clinical data generated in physiologically relevant pre-clinical models. Here we report the development of the chick embryo chorioallantoic membrane (CAM) model to study tumor growth and angiogenesis using breast cancer cell lines. MDA-MB-231 and MCF7 tumor cell lines were engrafted onto the chick embryo CAM to study tumor growth and treatment response. Tumor growth was evaluated through bioluminescence imaging and a significant increase in tumor size and vascularization was found over a 9-day period. We then evaluated the impact of anti-angiogenic drugs, axitinib and bevacizumab, on tumor growth and angiogenesis. Drug treatment significantly reduced tumor vascularization and size. Overall, our findings demonstrate that the chick embryo CAM is a clinically relevant model to monitor therapeutic response in breast cancer and can be used as a platform for drug screening to evaluate not only gross changes in tumor burden but physiological processes such as angiogenesis.

The different prognostic significance of polysialic acid and CD56 expression in tumor cells and lymphocytes identified in breast cancer.

Protein glycosylation, the attachment of carbohydrates onto proteins, is a fundamental process that alters the biological activity of proteins. Changes to glycosylation states are associated with many forms of cancer including breast cancer. Through immunohistological analysis of breast cancer patient tumors, we have discovered the expression of an atypical glycan-polysialic acid (polySia)-in breast cancer. Notably, we have identified polySia expression in not only tumor cells but also on tumor-infiltrating lymphocytes (TILs) and our study reveals ST8Sia4 as the predominant polysialyltransferase expressed. Evaluation of ST8Sia4 expression in tumor cells identified an association between high expression levels and poor patient outcomes whereas ST8Sia4 expression in infiltrating stromal cells was associated with good patient outcomes. Investigation into CD56, a protein known to be polysialylated, found CD56 and polySia expression on breast tumor cells and TILs. CD56 expression did not positively correlate with polySia expression except in patient tumors which expressed HER2. In these HER2 expressing tumors, CD56 expression was significantly associated with HER2 expression score. Evaluation of CD56 tumor cell expression identified a significant association between CD56 expression and poor patient outcomes. By contrast, CD56 expression on TILs was significantly associated with good clinical outcomes. Tumors with CD56+ TILs were also consistently polySia TIL positive. Interestingly, in tumors where TILs were CD56 low-to-negative, a polySia+ lymphocyte population was still identified and the presence of these lymphocytes was a poor prognostic indicator. Overall, this study provides the first detailed report of polySia and CD56 in breast cancer and demonstrates that the prognostic significance is dependent on the cell type expression within the tumor.

A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

Immunomodulatory Effects of Flavonoids: Possible Induction of T CD4+ Regulatory Cells Through Suppression of mTOR Pathway Signaling Activity.

The increasing rate of autoimmune disorders and cancer in recent years has been a controversial issue in all aspects of prevention, diagnosis, prognosis and treatment. Among dietary factors, flavonoids have specific immunomodulatory effects that might be of importance to several cancers. Over different types of immune cells, T lymphocytes play a critical role in protecting the immune system as well as in the pathogenesis of specific autoimmune diseases. One of the important mediators of metabolism and immune system is mTOR, especially in T lymphocytes. In the current review, we assessed the effects of flavonoids on the immune system and then their impact on the mTOR pathway. Flavonoids can suppress mTOR activity and are consequently able to induce the T regulatory subset.

Invadopodia are chemosensing protrusions that guide cancer cell extravasation to promote brain tropism in metastasis.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.