The major drawback of current anti-angiogenic therapies is drug resistance, mainly caused by overexpression of the transcription factor, hypoxia-inducible factor 1α (HIF-1α) as a result of treatment-induced hypoxia, which stimulates cancer cells to develop aggressive and immunosuppressive phenotypes. Moreover, the cancer cell resistance to anti-angiogenic therapies is deeply mediated by the communication between tumor cells and tumor-associated macrophages (TAMs)—the most important microenvironmental cells for the coordination of all supportive processes in tumor development. Thus, simultaneous targeting of TAMs and cancer cells could improve the outcome of the anti-angiogenic therapies. Since our previous studies proved that simvastatin (SIM) exerts strong antiproliferative actions on B16.F10 murine melanoma cells via reduction of TAMs-mediated oxidative stress and inhibition of intratumor production of HIF-1α, we investigated whether the antitumor efficacy of the anti-angiogenic agent—5,6-dimethylxanthenone-4-acetic acid (DMXAA) could be improved by its co-administration with the lipophilic statin. Our results provide confirmatory evidence for the ability of the combined treatment to suppress the aggressive phenotype of the B16.F10 melanoma cells co-cultured with TAMs under hypoxia-mimicking conditions in vitro. Thus, proliferation and migration capacity of the melanoma cells were strongly decelerated after the co-administration of SIM and DMXAA. Moreover, our data suggested that the anti-oxidant action of the combined treatment, as a result of melanogenesis stimulation, might be the principal cause for the simultaneous suppression of key molecules involved in melanoma cell aggressiveness, present in melanoma cells (HIF-1α) as well as in TAMs (arginase-1). Finally, the concomitant suppression of these proteins might have contributed to a very strong inhibition of the angiogenic capacity of the cell co-culture microenvironment.

An increasing number of studies published so far have evidenced the benefits of Simvastatin (SIM) and Doxorubicin (DOX) co-treatment in colorectal cancer. In view of this, the current study aimed to investigate the pharmaceutical development of liposomes co-encapsulating SIM and DOX, by implementing the Quality by Design (QbD) concept, as a means to enhance the antiproliferative effect of the co-formulation on C26 murine colon cancer cells co-cultured with macrophages. It is known that the quality profile of liposomes is dependent on the critical quality attributes (CQAs) of liposomes (drug entrapped concentration, encapsulation efficiency, size, zeta potential, and drug release profile), which are, in turn, directly influenced by various formulation factors and processing parameters. By using the design of experiments, it was possible to outline the increased variability of CQAs in relation to formulation factors and identify by means of statistical analysis the material attributes that are critical (phospholipids, DOX and SIM concentration) for the quality of the co-formulation. The in vitro studies performed on a murine colon cancer cell line highlighted the importance of delivering the optimal drug ratio at the target site, since the balance antiproliferative vs. pro-proliferative effects can easily be shifted when the molar ratio between DOX and SIM changes.

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Tailoring extracellular vesicles (EVs) as targeted drug delivery systems to enhance the therapeutic efficacy showed superior advantage over liposomal therapies. Herein, we developed a novel nanotool for targeting B16.F10 murine melanoma, based on EVs stabilized with Polyethylene glycol (PEG) and loaded with doxorubicin (DOX). Small EVs were efficiently enriched from melanoma cells cultured under metabolic stress by ultrafiltration coupled with size exclusion chromatography (UF-SEC) and characterized by size, morphology, and proteome. To reduce their clearance in vivo , EVs were PEGylated and passively loaded with DOX (PEG-EV-DOX). Our data suggested that the low PEG coverage of EVs might still favor EV surface protein interactions with target proteins from intratumor cells, ensuring their use as “Trojan horses” to deliver DOX to the tumor tissue. Moreover, our results showed a superior antitumor activity of PEG-EV-DOX in B16.F10 murine melanoma models in vivo compared to that exerted by clinically applied liposomal DOX in the same tumor model. The PEG-EV-DOX administration in vivo reduced NF-κB activation and increased BAX expression, suggesting better prognosis of EV-based therapy than liposomal DOX treatment. Collectively, our results highlight the promising potential of EVs as optimal tools for systemic delivery of DOX to solid tumors.

LCL-CURC-DOX demonstrated enhanced cytotoxicity on C26 murine colon cancer cells by inhibiting the production of the majority of factors involved in tumor-associated angiogenesis and inflammation and is now being evaluated in vivo regarding its efficacy towards tumor growth in colon cancer.