Meet me halfway: Are in vitro 3D cancer models on the way to replace in vivo models for nanomedicine development?

The complexity and diversity of the biochemical processes that occur during tumorigenesis and metastasis are frequently over-simplified in the traditional in vitro cell cultures. Two-dimensional cultures limit researchers' experimental observations and frequently give rise to misleading and contradictory results. Therefore, in order to overcome the limitations of in vitro studies and bridge the translational gap to in vivo applications, 3D models of cancer were developed in the last decades. The three dimensions of the tumor, including its cellular and extracellular microenvironment, are recreated by combining co-cultures of cancer and stromal cells in 3D hydrogel-based growth factors-inclusive scaffolds. More complex 3D cultures, containing functional blood vasculature, can integrate in the system external stimuli (e.g. oxygen and nutrient deprivation, cytokines, growth factors) along with drugs, or other therapeutic compounds. In this scenario, cell signaling pathways, metastatic cascade steps, cell differentiation and self-renewal, tumor-microenvironment interactions, and precision and personalized medicine, are among the wide range of biological applications that can be studied. Here, we discuss a broad variety of strategies exploited by scientists to create in vitro 3D cancer models that resemble as much as possible the biology and patho-physiology of in vivo tumors and predict faithfully the treatment outcome.

P-selectin axis plays a key role in microglia immunophenotype and glioblastoma progression.

Glioblastoma (GB) is a highly invasive type of brain cancer exhibiting poor prognosis. As such, its microenvironment plays a crucial role in its progression. Among the brain stromal cells, the microglia were shown to facilitate GB invasion and immunosuppression. However, the reciprocal mechanisms by which GB cells alter microglia/macrophages behavior are not fully understood. We propose that these mechanisms involve adhesion molecules such as the Selectins family. These proteins are involved in immune modulation and cancer immunity. We show that P-selectin mediates microglia-enhanced GB proliferation and invasion by altering microglia/macrophages activation state. We demonstrate these findings by pharmacological and molecular inhibition of P-selectin which leads to reduced tumor growth and increased survival in GB mouse models. Our work sheds light on tumor-associated microglia/macrophage function and the mechanisms by which GB cells suppress the immune system and invade the brain, paving the way to exploit P-selectin as a target for GB therapy.

Preclinical models and technologies to advance nanovaccine development.

The remarkable success of targeted immunotherapies is revolutionizing cancer treatment. However, tumor heterogeneity and low immunogenicity, in addition to several tumor-associated immunosuppression mechanisms are among the major factors that have precluded the success of cancer vaccines as targeted cancer immunotherapies. The exciting outcomes obtained in patients upon the injection of tumor-specific antigens and adjuvants intratumorally, reinvigorated interest in the use of nanotechnology to foster the delivery of vaccines to address cancer unmet needs. Thus, bridging nano-based vaccine platform development and predicted clinical outcomes the selection of the proper preclinical model will be fundamental. Preclinical models have revealed promising outcomes for cancer vaccines. However, only few cases were associated with clinical responses. This review addresses the major challenges related to the translation of cancer nano-based vaccines to the clinic, discussing the requirements for ex vivo and in vivo models of cancer to ensure the translation of preclinical success to patients.

Immune-mediated approaches against COVID-19.

The coronavirus disease-19 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The long incubation period of this new virus, which is mostly asymptomatic yet contagious, is a key reason for its rapid spread across the world. Currently, there is no worldwide-approved treatment for COVID-19. Therefore, the clinical and scientific communities have joint efforts to reduce the severe impact of the outbreak. Research on previous emerging infectious diseases have created valuable knowledge that is being exploited for drug repurposing and accelerated vaccine development. Nevertheless, it is important to generate knowledge on SARS-CoV-2 mechanisms of infection and its impact on host immunity, to guide the design of COVID-19 specific therapeutics and vaccines suitable for mass immunization. Nanoscale delivery systems are expected to play a paramount role in the success of these prophylactic and therapeutic approaches. This Review provides an overview of SARS-CoV-2 pathogenesis and examines immune-mediated approaches currently explored for COVID-19 treatments, with an emphasis on nanotechnological tools.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

Tailoring the lipid composition of nanoparticles modulates their cellular uptake and affects the viability of triple negative breast cancer cells.

Lipid nanoparticles are used widely as anticancer drug and gene delivery systems. Internalizing into the target cell is a prerequisite for the proper activity of many nanoparticulate drugs. We show here, that the lipid composition of a nanoparticle affects its ability to internalize into triple-negative breast cancer cells. The lipid headgroup had the greatest effect on enhancing cellular uptake compared to other segments of the molecule. Having a receptor-targeted headgroup induced the greatest increase in cellular uptake, followed by cationic amine headgroups, both being superior to neutral (zwitterion) phosphatidylcholine or to negatively-charged headgroups. The lipid tails also affected the magnitude of cellular uptake. Longer acyl chains facilitated greater liposomal cellular uptake compared to shorter tails, 18:0 > 16:0 > 14:0. When having the same lipid tail length, unsaturated lipids were superior to saturated ones, 18:1 > 18:0. Interestingly, liposomes composed of phospholipids having 14:0 or 12:0-carbon-long-tails, such as DMPC and DLPC, decreased cell viability in a concertation dependent manner, due to a destabilizing effect these lipids had on the cancer cell membrane. Contrarily, liposomes composed of phospholipids having longer carbon tails (16:0 and 18:0), such as DPPC and HSPC, enhanced cancer cell proliferation. This effect is attributed to the integration of the exogenous liposomal lipids into the cancer-cell membrane, supporting the proliferation process. Cholesterol is a common lipid additive in nanoscale formulations, rigidifying the membrane and stabilizing its structure. Liposomes composed of DMPC (14:0) showed increased cellular uptake when enriched with cholesterol, both by endocytosis and by fusion. Contrarily, the effect of cholesterol on HSPC (18:0) liposomal uptake was minimal. Furthermore, the concentration of nanoparticles in solution affected their cellular uptake. The higher the concentration of nanoparticles the greater the absolute number of nanoparticles taken up per cell. However, the efficiency of nanoparticle uptake, i.e. the percent of nanoparticles taken up by cells, decreased as the concentration of nanoparticles increased. This study demonstrates that tuning the lipid composition and concentration of nanoscale drug delivery systems can be leveraged to modulate their cellular uptake.