CD133+ ovarian cancer stem-like cells promote non-stem cancer cell metastasis via CCL5 induced epithelial-mesenchymal transition

Cancer stem cells (CSCs, also called cancer stem-like cells, CSLCs) can function as “seed cells” for tumor recurrence and metastasis. Here, we report that, in the presence of CD133⁺ ovarian CSLCs, CD133⁻ non-CSLCs can undergo an epithelial-mesenchymal transition (EMT)-like process and display enhanced metastatic capacity in vitro and in vivo. Highly elevated expression of chemokine (C-C motif) ligand 5 (CCL5) and its receptors chemokine (C-C motif) receptor (CCR) 1/3/5 are observed in clinical and murine metastatic tumor tissues from epithelial ovarian carcinomas. Mechanistically, paracrine CCL5 from ovarian CSLCs activates the NF-κB signaling pathway in ovarian non-CSLCs via binding CCR1/3/5, thereby inducing EMT and tumor invasion. Taken together, our results redefine the metastatic potential of non-stem cancer cells and provide evidence that targeting the CCL5:CCR1/3/5-NF-κB pathway could be an effective strategy to prevent ovarian cancer metastasis.     

Antibiotics that target mitochondria effectively eradicate cancer stem cells,across multiple tumor types: Treating cancer like an infectious disease

Here, we propose a new strategy for the treatment of early cancerous lesions and advanced metastatic disease, via the selective targeting of cancer stem cells (CSCs), a.k.a., tumor-initiating cells (TICs). We searched for a global phenotypic characteristic that was highly conserved among cancer stem cells, across multiple tumor types, to provide a mutation-independent approach to cancer therapy. This would allow us to target cancer stem cells, effectively treating cancer as a single disease of “stemness”, independently of the tumor tissue type. Using this approach, we identified a conserved phenotypic weak point – a strict dependence on mitochondrial biogenesis for the clonal expansion and survival of cancer stem cells. Interestingly, several classes of FDA-approved antibiotics inhibit mitochondrial biogenesis as a known “side-effect”, which could be harnessed instead as a “therapeutic effect”. Based on this analysis, we now show that 4-to-5 different classes of FDA-approved drugs can be used to eradicate cancer stem cells, in 12 different cancer cell lines, across 8 different tumor types (breast, DCIS, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma (brain)). These five classes of mitochondrially-targeted antibiotics include: the erythromycins, the tetracyclines, the glycylcyclines, an anti-parasitic drug, and chloramphenicol. Functional data are presented for one antibiotic in each drug class: azithromycin, doxycycline, tigecycline, pyrvinium pamoate, as well as chloramphenicol, as proof-of-concept. Importantly, many of these drugs are non-toxic for normal cells, likely reducing the side effects of anti-cancer therapy. Thus, we now propose to treat cancer like an infectious disease, by repurposing FDA-approved antibiotics for anti-cancer therapy, across multiple tumor types. These drug classes should also be considered for prevention studies, specifically focused on the prevention of tumor recurrence and distant metastasis. Finally, recent clinical trials with doxycycline and azithromycin (intended to target cancer-associated infections, but not cancer cells) have already shown positive therapeutic effects in cancer patients, although their ability to eradicate cancer stem cells was not yet appreciated.     

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a “stemness and metastatic” signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.     

On the origin and destination of cancer stem cells: a conceptual evaluation

Despite remaining uncertainties and ongoing research it is possible to draw up a model for the role of (cancer) stem cells in both the initiation and progression of cancer towards metastasis. The cancer stem cell of origin and the cancer stem cell are, despite phenotypic similarities, genotypically different entities. Given the right circumstances provided by a combination of genomic changes and biochemical and physical interactions with its microenvironment, an epithelial cancer cell may undergo a phenotypic epithelial mesenchymal transition (EMT) towards a cancer stem cell. This transition conveys upon the cell crucial stem cell-like abilities which facilitate migration into the blood circulation as an individual circulating tumor cell, survive there, and subsequently seed into organ tissue where, once more in close interaction with its microenvironment, the process of clonal self renewal may start, leading to a metastatic tumor. Both in the primary tumor as well as in the metastatic tumor, partial differentiation of the cancer stem cell progeny leads to phenotypic heterogeneity. Throughout this complex process of cancer metastasis similarities with the way stem cells function during embryonic development, including the signaling pathways that mediate these functions, are evident. Deeper insight in the EMT process, plasticity of the resulting cancer stem cells, and the role of cancer stem cells in the metastatic process is expected to lead to novel anti-metastatic cancer therapies. Emerging human in vitro cancer models in the form of “organ-on-a-chip” may contribute valuable novel research tools to achieve this aim.     

Epidermal growth factor receptor mutation mediates cross-resistance to panitumumab and cetuximab in gastrointestinal cancer

Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/− EGFR antibodies by next-generation sequencing (“tumor tissue” cohort). We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab. The EGFR G465R mutation introduces a positive charge within the overlap of the panitumumab and cetuximab epitopes. It abrogates antibody binding and mediates cross-resistance to both antibodies in EGFR G465R-transfected Ba/F3 cells. In circulating tumor DNA from an independent “liquid biopsy” cohort of 27 patients, we found this novel mutation in 1 out of 6 panitumumab-treated cases while about one third of patients show acquired RAS mutations. We show that acquired resistance by epitope-changing mutations also emerges during panitumumab treatment, which can be easily detected by a liquid biopsy approach even before clinical resistance occurs and this may help in tailoring EGFR-targeted therapies.     

Approaches to isolation and molecular characterization of disseminated tumor cells

Micrometastatic cells in the bone marrow, now usually referred to as “disseminated tumor cells (DTCs)”, can be detected in early stage cancer patients. It has been hypothesized that DTCs represent key intermediates in the metastatic process as possible precursors of bone and visceral metastases, and are indicators of metastatic potential. Indeed, multiple clinical studies have unequivocally demonstrated the prognostic value of these cells in breast and other cancers, as DTCs have been associated with adverse outcomes, including inferior overall and disease-free survival. Despite this established clinical significance, the molecular nature of DTCs remains elusive. The complexity of the bone marrow poses a unique challenge in the isolation and direct characterization of these rare cells. However, recent advances in rare-cell technology along with technical improvements in analyzing limited cell inputs have enabled the molecular profiling of DTCs. In this review, we discuss research featuring the isolation and genomic analysis of DTCs. Emerging work on the molecular characterization of DTCs is now providing new insights into the biology of these cells.     

Anticancer activity of halofuginone in a preclinical model of osteosarcoma: inhibition of tumor growth and lung metastases

Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because we recently demonstrated that TGF-β/Smad cascade plays a crucial role in osteosarcoma metastatic progression, we investigated the effect of halofuginone, identified as an inhibitor of the TGF-β/Smad3 cascade, on osteosarcoma progression. A preclinical model of osteosarcoma was used to evaluate the impact of halofuginone on tumor growth, tumor microenvironment and metastasis development. In vivo experiments showed that halofuginone reduces primary tumor growth and lung metastases development. In vitro experiments demonstrated that halofuginone decreases cell viability mainly by its ability to induce caspase-3 dependent cell apoptosis. Moreover, halofuginone inhibits the TGF-β/Smad3 cascade and the response of TGF-β key targets involved in the metastases dissemination process such as MMP-2. In addition, halofuginone treatment affects the “vicious cycle” established between tumor and bone cells, and therefore the tumor-associated bone osteolysis. Together, these results demonstrate that halofuginone decreased primary osteosarcoma development and associated lung metastases by targeting both the tumor cells and the tumor microenvironment. Using halofuginone may be a promising therapeutic strategy against tumor progression of osteosarcoma specifically against lung metastases dissemination.     

Lysyl hydroxylase 2-induced collagen cross-link switching promotes metastasis in head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide and incidence rates are continuing to rise globally. HNSCC patient prognosis is closely related to the occurrence of tumor metastases, and collagen within the tumor microenvironment (TME) plays a key role in this process. Lysyl hydroxylase 2 (LH2), encoded by the Procollagen-Lysine,2-Oxoglutarate 5-Dioxygenase 2 (PLOD2) gene, catalyzes hydroxylation of telopeptidyl lysine (Lys) residues of fibrillar collagens which then undergo subsequent modifications to form stable intermolecular cross-links that change the biomechanical properties (i.e. quality) of the TME. While LH2-catalyzed collagen modification has been implicated in driving tumor progression and metastasis in diverse cancers, little is known about its role in HNSCC progression. Thus, using gain- and loss-of-function studies, we examined the effects of LH2 expression levels on collagen cross-linking and cell behavior in vitro and in vivo using a tractable bioluminescent imaging-based orthotopic xenograft model. We found that LH2 overexpression dramatically increases HNSCC cell migratory and invasive abilities in vitro and that LH2-driven changes in collagen cross-linking robustly induces metastasis in vivo. Specifically, the amount of LH2-mediated collagen cross-links increased significantly with PLOD2 overexpression, without affecting the total quantity of collagen cross-links. Conversely, LH2 knockdown significantly blunted HNSCC cells invasive capacity in vitro and metastatic potential in vivo. Thus, regardless of the total “quantity” of collagen crosslinks, it is the “quality” of these cross-links that is the key driver of HNSCC tumor metastatic dissemination. These data implicate LH2 as a key regulator of HNSCC tumor invasion and metastasis by modulating collagen cross-link quality and suggest that therapeutic strategies targeting LH2-mediated collagen cross-linking in the TME may be effective in controlling tumor progression and improving disease outcomes.     

Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry,enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts

Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by “vasculogenic” or “vascular” mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2. We show that melanoma cells lose their ability to form tubule-like structures in vitro after blocking syndecan-1 activity by the specific human recombinant antibody, OC-46F2. Moreover, in a human melanoma xenograft model, the combined therapy using OC-46F2 and L19-IL2, an immunocytokine specific for the tumor angiogenic-associated B-fibronectin isoform(B-FN), led to a complete inhibition of tumor growth until day 90 from tumor implantation in 71% of treated mice, with statistically significant differences compared to groups treated with OC-46F2 or L19-IL2 as monotherapy. Furthermore, in the tumors recovered from mice treated with OC-46F2 either as monotherapy or in combination with L19-IL2, we observed a dramatic decrease of vascular density and loss of VM structures. These findings indicate for the first time a role of syndecan-1 in melanoma VM and that targeting syndecan-1, together with B-FN, could be promising in improving the treatment of metastatic melanoma.     

Monocytes and macrophages,implications for breast cancer migration and stem cell-like activity and treatment

Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with poor prognosis and high grade tumours we used the THP-1 cell line to model monocyte-macrophage differentiation in co-culture with four breast cancer cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468) to model in vivo cellular interactions. Polarisation into M1 and M2 subtypes was confirmed by specific cell marker expression of ROS and HLA-DR, respectively. Co-culture with all types of macrophage increased migration of ER-positive breast cancer cell lines, while M2-macrophages increased mammosphere formation, compared to M1-macrophages, in all breast cancer cells lines. Treatment of cells with Zoledronate in co-culture reduced the “pro-tumourigenic” effects (increased mammospheres/migration) exerted by macrophages. Direct treatment of breast cancer cells in homotypic culture was unable to reduce migration or mammosphere formation.Macrophages promote “pro-tumourigenic” cellular characteristics of breast cancer cell migration and stem cell activity. Zoledronate targets macrophages within the microenvironment which in turn, reduces the “pro-tumourigenic” characteristics of breast cancer cells. Zoledronate offers an exciting new treatment strategy for both primary and metastatic breast cancer.     

Consensus recommendations for MRI and PET imaging of primary central nervous system lymphoma: guideline statement from the International Primary CNS Lymphoma Collaborative Group (IPCG)

Advanced molecular and pathophysiologic characterization of primary central nervous system lymphoma (PCNSL) has revealed insights into promising targeted therapeutic approaches. Medical imaging plays a fundamental role in PCNSL diagnosis, staging, and response assessment. Institutional imaging variation and inconsistent clinical trial reporting diminishes the reliability and reproducibility of clinical response assessment. In this context, we aimed to: (1) critically review the use of advanced positron emission tomography (PET) and magnetic resonance imaging (MRI) in the setting of PCNSL; (2) provide results from an international survey of clinical sites describing the current practices for routine and advanced imaging, and (3) provide biologically based recommendations from the International PCNSL Collaborative Group (IPCG) on adaptation of standardized imaging practices. The IPCG provides PET and MRI consensus recommendations built upon previous recommendations for standardized brain tumor imaging protocols (BTIP) in primary and metastatic disease. A biologically integrated approach is provided to addresses the unique challenges associated with the imaging assessment of PCNSL. Detailed imaging parameters facilitate the adoption of these recommendations by researchers and clinicians. To enhance clinical feasibility, we have developed both “ideal” and “minimum standard” protocols at 3T and 1.5T MR systems that will facilitate widespread adoption.     

Dynamic proteomic change of tumor and immune organs in an immune-competent hepatocellular carcinoma mouse model

Subcutaneous implantation of a human cancer cell line in immune-deficient mice (CDX) is a commonly used tool in preclinical studies for the assessment of potential anti-cancer drugs. As immunotherapy is transforming cancer treatment, tumor models in immunocompetent mice are necessary for us to understand the immune aspects of tumor biology. However, the systemic immune response to the implantation of cancer cells at proteome level is unclear. In this study, we characterized the dynamic proteomic changes of subcutaneous tumors and 5 immune organs (draining lymph node, mesenteric lymph node, spleen, thymus and marrow) at six time points after implantation using a Hepa1-6 derived allograft mouse model. Our data suggest that interaction of the implanted tumor cells with mouse immune system followed the trajectory of “tumor rejection” to “immune evasion” in that the tumor gained the ability to evade the immune system for growth. Furthermore, anti-PDL2 antibody was validated here as an optional immunotherapy strategy to inhibit the growth of Hepa1-6 subcutaneous tumors. These findings from our study provided valuable information for the understanding of tumor and immune interaction and shed light on the rational design for clinical cancer treatment and other preclinical experiments.     

EphA2 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes

EphA2 (Eck) is a tyrosine kinase receptor that is overexpressed in several human cancers such as breast, colon, lung, prostate, gastric carcinoma, and metastatic melanoma but not in nonmalignant counterparts. To validate EphA2 as a tumor antigen recognized by CD8+ T lymphocytes, we used reverse immunology approach to identify HLA-A*0201-restricted epitopes. Peptides bearing the HLA-A*0201-specific anchor motifs were analyzed for their capacity to bind and stabilize the HLA-A*0201 molecules. Two peptides, EphA2(58) and EphA2(550), with a high affinity for HLA-A*0201 were selected. Both peptides were immunogenic in the HLA-A*0201-transgenic HHD mice. Interestingly, peptide-specific murine CTLs cell lines responded to COS-7 cells coexpressing HLA-A*0201 and EphA2 and to EphA2-positive human tumor cells of various origin (renal cell, lung, and colon carcinoma and sarcoma). This demonstrates that EphA2(58) and EphA2(550) are naturally processed from endogenous EphA2. In addition, EphA2(58) and EphA2(550) stimulated specific CD8(+) T cells from healthy donor peripheral blood mononuclear cells. These T cells recognized EphA2-positive human tumor cells in an HLA-A*0201-restricted manner. Interestingly, EphA2-specific CD8+ T cells were detected in the peripheral blood mononuclear cells of prostate cancer patients. These results show for the first time that EphA2 is a tumor rejection antigen and lead us to propose EphA2(58) and EphA2(550) peptides for a broad-spectrum-tumor immunotherapy.     

Breast cancer metastasis: immune profiling of lymph nodes reveals exhaustion of effector T cells and immunosuppression

Sentinel lymph nodes are the first nodes draining the lymph from a breast and could reveal early changes in the host immune system upon dissemination of breast cancer cells. To investigate this, we performed single‐cell immune profiling of lymph nodes with and without metastatic cells. Whereas no significant changes were observed for B‐cell and natural killer (NK)‐cell subsets, metastatic lymph nodes had a significantly increased frequency of CD8 T cells and a skewing toward an effector/memory phenotype of CD4 and CD8 T cells, suggesting an ongoing immune response. Additionally, metastatic lymph nodes had an increased frequency of TIGIT (T‐cell immunoreceptor with Ig and ITIM domains)‐positive T cells with suppressed TCR signaling compared with non‐metastatic nodes, indicating exhaustion of effector T cells, and an increased frequency of regulatory T cells (Tregs) with an activated phenotype. T‐cell alterations correlated with the percentage of metastatic tumor cells, reflecting the presence of metastatic tumor cells driving T effector cells toward exhaustion and promoting immunosuppression by recruitment or increased differentiation toward Tregs. These results show that immune suppression occurs already in early stages of tumor progression.     

Tumor‐targeted fluorescence labeling systems for cancer diagnosis and treatment

Conventional imaging techniques are available for clinical identification of tumor sites. However, detecting metastatic tumor cells that are spreading from primary tumor sites using conventional imaging techniques remains difficult. In contrast, fluorescence‐based labeling systems are useful tools for detecting tumor cells at the single‐cell level in cancer research. The ability to detect fluorescent‐labeled tumor cells enables investigations of the biodistribution of tumor cells for the diagnosis and treatment of cancer. For example, the presence of fluorescent tumor cells in the peripheral blood of cancer patients is a predictive biomarker for early diagnosis of distant metastasis. The elimination of fluorescent tumor cells without damaging normal tissues is ideal for minimally invasive treatment of cancer. To capture fluorescent tumor cells within normal tissues, however, tumor‐specific activated target molecules are needed. This review focuses on recent advances in tumor‐targeted fluorescence labeling systems, in which indirect reporter labeling using tumor‐specific promoters is applied to fluorescence labeling of tumor cells for the diagnosis and treatment of cancer. Telomerase promoter‐dependent fluorescence labeling using replication‐competent viral vectors produces fluorescent proteins that can be used to detect and eliminate telomerase‐positive tumor cells. Tissue‐specific promoter‐dependent fluorescence labeling enables identification of specific tumor cells. Vimentin promoter‐dependent fluorescence labeling is a useful tool for identifying tumor cells that undergo epithelial–mesenchymal transition (EMT). The evaluation of tumor cells undergoing EMT is important for accurately assessing metastatic potential. Thus, tumor‐targeted fluorescence labeling systems represent novel platforms that enable the capture of tumor cells for the diagnosis and treatment of cancer.     

Detecting cell-in-cell structures in human tumor samples by E-cadherin/CD68/CD45 triple staining

Although Cell-in-cell structures (CICs) had been documented in human tumors for decades, it is unclear what types of CICs were formed largely due to low resolution of traditional way such as H&E staining. In this work, we employed immunofluorescent method to stain a panel of human tumor samples simultaneously with antibodies against E-cadherin for Epithelium, CD68 for Macrophage and CD45 for Leukocytes, which we termed as “EML method” based on the cells detected. Detail analysis revealed four types of CICs, with tumor cells or macrophage engulfing tumor cells or leukocytes respectively. Interestingly, tumor cells seem to be dominant over macrophage (93% vs 7%) as the engulfer cells in all CICs detected, whereas the overall amount of internalized tumor cells is comparable to that of internalized CD45⁺ leukocytes (57% vs 43%). The CICs profiles vary from tumor to tumor, which may indicate different malignant stages and/or inflammatory conditions. Given the potential impacts different types of CICs might have on tumor growth, we therefore recommend EML analysis of tumor samples to clarify the correlation of CICs subtypes with clinical prognosis in future researches.     

MiR-19b-3p and miR-101-3p as potential biomarkers for prostate cancer diagnosis and prognosis

Prostate cancer (PCa) is the most commonly diagnosed male malignancy worldwide. Early diagnosis and metastases detection are crucial features to diminish patient mortality. High fat diet (HFD) and metabolic syndrome increase PCa risk and aggressiveness. Our goal was to identify miRNAs-based biomarkers for PCa diagnosis and prognosis associated with HFD. Mice chronically fed with a HFD or control diet (CD) were subcutaneously inoculated with androgen insensitive PC3 cells. Xenografts from HFD-fed mice showed increased expression of 7 miRNAs that we named “candidates” compared to CD-fed mice. These miRNAs modulate specific metabolic and cancer related pathways. Using bioinformatic tools and human datasets we found that hsa-miR-19b-3p and miR-101-3p showed more than 1,100 validated targets involved in proteoglycans in cancer and fatty acid biosynthesis. These miRNAs were significantly increased in the bloodstream of PCa patients compared to non-PCa volunteers, and in prostate tumors compared to normal adjacent tissues (NAT). Interestingly, both miRNAs were also increased in tumors of metastatic patients compared to tumors of non-metastatic patients. Further receiver-operating characteristic (ROC) analysis determined that hsa-miR-19b-3p and hsa-miR-101-3p in serum showed poor predictive power to discriminate PCa from non-PCa patients. Hsa-miR-19b-3p showed the best score to discriminate between tumor and NAT, while hsa-miR-101-3p was useful to differentiate between metastatic and non-metastatic PCa patients. Hsa-miR-101-3p was increased in exosomes isolated from blood of PCa patients. Although more detailed functional exploration and validation of the molecular mechanisms are required, we identified hsa-miR-19b-3p and hsa-miR-101-3p with high potential for PCa diagnosis and prognosis.     

Anti-cancer potency of tasquinimod is enhanced via albumin-binding facilitating increased uptake in the tumor microenvironment

Tasquinimod, an orally active quinoline-3-carboxamide, binds with high affinity to HDAC4 and S100A9 in cancer and infiltrating host cells within compromised tumor microenvironment inhibiting adaptive survival pathways needed for an angiogenic response. Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod''s ability to inhibit endothelial “sprouting” in vitro or anti-cancer efficacy against human prostate cancer xenografts in vivo.Tasquinimod''s potency is facilitated by its reversible binding (K_d < 35 μM) to the IIA subdomain of albumin (Sudlow''s site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the “enhanced permeability and retention” (i.e., EPR) effect. Thus, despite plasma levels of < 1 μM, the EPR effect results in intracellular drug concentrations of 2-3 μM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC₅₀ ~ 0.5 μM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.