Giulia Golinelli

Cancer treatment, particularly chemotherapy, requires balancing efficacy and toxicity. Although traditional monitoring methods can lead to suboptimal outcomes, emerging implantable chemical sensors can complement them by providing precise, real-time drug monitoring at tumor sites, although the technology remains in its early stages. Here, we introduce an ultrathin, bioresorbable implantable biosensor for real-time doxorubicin monitoring in vivo with high spatiotemporal resolution. The sensor amplifies the drug’s fluorescence, enabling successful tracking of doxorubicin through the skin in live mice following intravenous injection. When paired with a reusable electronic patch, the biosensor facilitates seamless data collection and wireless transmission. A 3-month biocompatibility study, including systemic toxicity assessments, histological and blood analyses, confirms complete biodegradation with no observed toxicity. By directly measuring chemotherapeutic drug levels in tissues over time, our sensor enhances traditional monitoring methods, enabling clinicians to optimize dosing during cancer treatment and reduce the risk of locoregional recurrence following tumor removal.

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients.

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVO^Ⓡ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing’s sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVO^Ⓡ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVO^Ⓡ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase.

Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and ¹⁸F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.

Since mesenchymal stromal/stem cells (MSCs) were discovered, researchers have been drawn to study their peculiar biological features, including their immune privileged status and their capacity to selectively migrate into inflammatory areas, including tumors. These properties make MSCs promising cellular vehicles for the delivery of therapeutic molecules in the clinical setting. In recent decades, the engineering of MSCs into biological vehicles carrying anticancer compounds has been achieved in different ways, including the loading of MSCs with chemotherapeutics or drug functionalized nanoparticles (NPs), genetic modifications to force the production of anticancer proteins, and the use of oncolytic viruses. Recently, it has been demonstrated that wild-type and engineered MSCs can release extracellular vesicles (EVs) that contain therapeutic agents. Despite the enthusiasm for MSCs as cyto-pharmaceutical agents, many challenges, including controlling the fate of MSCs after administration, must still be considered. Preclinical results demonstrated that MSCs accumulate in lung, liver, and spleen, which could prevent their engraftment into tumor sites. For this reason, physical, physiological, and biological methods have been implemented to increase MSC concentration in the target tumors. Currently, there are more than 900 registered clinical trials using MSCs. Only a small fraction of these are investigating MSC-based therapies for cancer, but the number of these clinical trials is expected to increase as technology and our understanding of MSCs improve. This review will summarize MSC-based antitumor therapies to generate an increasing awareness of their potential and limits to accelerate their clinical translation.

Background: Adipose tissue derived mesenchymal stromal/stem cells (ASC) can be expanded using supernatant rich in growth factors (SRGF) as Good Manufacturing Practice compatible additive, instead of fetal bovine serum (FBS). After transendothelial migration, ASC can migrate to cancer masses where they can release active substances. Due to their homing and secretion properties ASC can be used as targeted drug delivery vehicles. Nevertheless, the fraction of ASC actually reaching the tumor target is limited. The impact of culture conditions on ASC homing potential on cancer cells is unknown. Methods: In dynamic in vitro conditions, we perfused FBS or SRGF ASC in flow chambers coated with collagen type I and fibronectin or seeded with endothelial cells or with HT1080, T98G and Huh7 cancer cells. Expression of selected adhesion molecules was evaluated by standard cytofluorimetry. Dynamic intracellular calcium concentration changes were evaluated in microfluidic and static conditions. Results: When compared to FBS ASC, not specific adhesion of SRGF ASC on collagen type I and fibronectin was lower (−33.9%±12.2% and −45.3%±16.9%), while on-target binding on HT1080 and T98G was enhanced (+147%±8% and 120.5%±5.2%). Adhesion of both FBS and SRGF ASC on Huh7 cells was negligible. As confirmed by citofluorimetry and by function-blocking antibody, SRGF mediated decrease of CD49a expression accounted for lower SRGF-ASC avidity for matrix proteins. Upon stimulation with calcium ionophore in static conditions, mobilization of intracellular calcium in SRGF ASC was greater than in FBS ASC. In dynamic conditions, upon adhesion on matrix proteins and HT1080 cells, SRGF ASC showed marked oscillatory calcium concentration changes. Conclusions: SRGF can enhance specific ASC binding capacity on selected cancer cells as HT1080 (fibrosarcoma) and T98G (glioblastoma) cells. Upon cell-cell adhesion, SRGF ASC activate intracellular responses potentially improving cell secretion functions. SRGF ASC could be considered as suitable drug delivery vehicle for cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset.