Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry.

Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.

Advances in the discovery and development of melanoma drug therapies.

INTRODUCTION: Therapeutic strategies for melanoma have evolved significantly over the last decade shifting from cytotoxic chemotherapies like dacarbazine to targeted therapies and immunotherapies including immune checkpoint inhibitors. These new drug therapies have improved overall as well as progression-free survival, lowering the mortality of this cancer for melanoma patients with advanced disease. Newer strategies incorporate combination therapies that harness synergies between mechanisms of anticancer efficacy as well as help overcome resistance issues of monotherapies, which remain a challenge. AREAS COVERED: This review looks at each class of drug therapy for melanoma and provides an overview of the preclinical mechanism of action, the clinical efficacy data, and their applications in combination therapy regimens. NCCN treatment guidelines, safety, toxicity, and immune-related adverse events are also described as well as a note on cost. EXPERT OPINION: Numerous ongoing trials continue to evaluate the role of novel therapies and combinations for this challenging disease and understanding their mechanism of action, risks, benefits, and treatment guidelines can help care providers and patients have a more comprehensive and tailored discussion of treatment options and expectations.

A novel C-terminal Hsp90 inhibitor KU758 synergizes efficacy in combination with BRAF or MEK inhibitors and targets drug-resistant pathways in BRAF-mutant melanomas.

Melanoma remains the most aggressive and fatal form of skin cancer, despite several FDA-approved targeted chemotherapies and immunotherapies for use in advanced disease. Of the 100 350 new patients diagnosed with melanoma in 2020 in the US, more than half will develop metastatic disease leading to a 5-year survival rate <30%, with a majority of these developing drug-resistance within the first year of treatment. These statistics underscore the critical need in the field to develop more durable therapeutics as well as those that can overcome chemotherapy-induced drug resistance from currently approved agents. Fortunately, several of the drug-resistance pathways in melanoma, including the proteins in those pathways, rely in part on Hsp90 chaperone function. This presents a unique and novel opportunity to simultaneously target multiple proteins and drug-resistant pathways in this disease via molecular chaperone inhibition. Taken together, we hypothesize that our novel C-terminal Hsp90 inhibitor, KU758, in combination with the current standard of care targeted therapies (e.g. vemurafenib and cobimetinib) can both synergize melanoma treatment efficacy in BRAF-mutant tumors, as well as target and overcome several major resistance pathways in this disease. Using in vitro proliferation and protein-based Western Blot analyses, our novel inhibitor, KU758, potently inhibited melanoma cell proliferation (without induction of the heat shock response) in vitro and synergized with both BRAF and MEK inhibitors in inhibition of cell migration and protein expression from resistance pathways. Overall, our work provides early support for further translation of C-terminal Hsp90 inhibitor and mitogen-activated protein kinase pathway inhibitor combinations as a novel therapeutic strategy for BRAF-mutant melanomas.

Metabolic engineering generates a transgene-free safety switch for cell therapy.

Safeguard mechanisms can ameliorate the potential risks associated with cell therapies but currently rely on the introduction of transgenes. This limits their application owing to immunogenicity or transgene silencing. We aimed to create a control mechanism for human cells that is not mediated by a transgene. Using genome editing methods, we disrupt uridine monophosphate synthetase (UMPS) in the pyrimidine de novo synthesis pathway in cell lines, pluripotent cells and primary human T cells. We show that this makes proliferation dependent on external uridine and enables us to control cell growth by modulating the uridine supply, both in vitro and in vivo after transplantation in xenograft models. Additionally, disrupting this pathway creates resistance to 5-fluoroorotic acid, which enables positive selection of UMPS-knockout cells. We envision that this approach will add an additional level of safety to cell therapies and therefore enable the development of approaches with higher risks, especially those that are intended for limited treatment durations.

Fibronectin-Expressing Mesenchymal Tumor Cells Promote Breast Cancer Metastasis.

Tumor metastasis is connected to epithelial-mesenchymal heterogeneity (EMH) and the extracellular matrix (ECM) within the tumor microenvironment. Mesenchymal-like fibronectin (FN) expressing tumor cells enhance metastasis within tumors that have EMH. However, the secondary tumors are primarily composed of the FN null population. Interestingly, during tumor cell dissemination, the invasive front has more mesenchymal-like characteristics, although the outgrowths of metastatic colonies consist of a more epithelial-like population of cells. We hypothesize that soluble FN provided by mesenchymal-like tumor cells plays a role in supporting the survival of the more epithelial-like tumor cells within the metastatic niche in a paracrine manner. Furthermore, due to a lower rate of proliferation, the mesenchymal-like tumor cells become a minority population within the metastatic niche. In this study, we utilized a multi-parametric cell-tracking algorithm and immunoblotting to evaluate the effect of EMH on the growth and invasion of an isogenic cell series within a 3D collagen network using a microfluidic platform. Using the MCF10A progression series, we demonstrated that co-culture with FN-expressing MCF10CA1h cells significantly enhanced the survival of the more epithelial MCF10CA1a cells, with a two-fold increase in the population after 5 days in co-culture, whereas the population of the MCF10CA1a cells began to decrease after 2.5 days when cultured alone (p < 0.001). However, co-culture did not significantly alter the rate of proliferation for the more mesenchymal MCF10CA1h cells. Epithelial tumor cells not only showed prolonged survival, but migrated significantly longer distances (350 µm compared with 150 µm, respectively, p < 0.01) and with greater velocity magnitude (4.5 µm/h compared with 2.1 µm/h, respectively, p < 0.001) under co-culture conditions and in response to exogenously administered FN. Genetic depletion of FN from the MCF10CA1h cells resulted in a loss of survival and migration capacity of the epithelial and mesenchymal populations. These data suggest that mesenchymal tumor cells may function to support the survival and outgrowth of more epithelial tumor cells within the metastatic niche and that inhibition of FN production may provide a valuable target for treating metastatic disease.

The Dynamic Relationship of Breast Cancer Cells and Fibroblasts in Fibronectin Accumulation at Primary and Metastatic Tumor Sites.

In breast cancer (BC), tissue stiffening via fibronectin (FN) and collagen accumulation is associated with advanced disease progression at both the primary tumor and metastatic sites. Here, we evaluate FN production in 15 BC cell lines, representing a variety of subtypes, phenotypes, metastatic potentials, and chemotherapeutic sensitivities. We demonstrate that intracellular and soluble FN is initially lost during tumorigenic transformation but is rescued in all lines with epithelial-mesenchymal plasticity (EMP). Importantly, we establish that no BC cell line was able to independently organize a robust FN matrix. Non-transformed mammary epithelial cells were also unable to deposit FN matrices unless transglutaminase 2, a FN crosslinking enzyme, was overexpressed. Instead, BC cells manipulated the FN matrix production of fibroblasts in a phenotypic-dependent manner. In addition, varied accumulation levels were seen depending if the fibroblasts were conditioned to model paracrine signaling or endocrine signaling of the metastatic niche. In the former, fibroblasts conditioned by BC cultures with high EMP resulted in the largest FN matrix accumulation. In contrast, mesenchymal BC cells produced extracellular vesicles (EV) that resulted in the highest levels of matrix formation by conditioned fibroblasts. Overall, we demonstrate a dynamic relationship between tumor and stromal cells within the tumor microenvironment, in which the levels and fibrillarization of FN in the extracellular matrix are modulated during the particular stages of disease progression.

Correlation of Structural and Macroscopic Properties of Starches with Their Tabletability Using the SM(2) Approach.

The effects of PURE-DENT® and SPRESS® starch properties on their compression behavior was characterized using "SM(2) " approach (structural properties, macroscopic properties, and multivariate analysis). Moisture sorption rate constants, moisture content, amylose and amylopectin degradation enthalpy, percent crystallinity, amylose-amylopectin ratio, and cross-linking degree were used to profile starch structural properties. Particle density, particle size distribution, and Heckel compression descriptors [yield pressure (YP) of plastic deformation, and elastic recovery] were used as macroscopic descriptors. The structural and macroscopic properties were correlated qualitatively [principal component analysis (PCA)] and quantitatively [standard least square regression (SLSR)] with the tablet mechanical strength (TMS). These analyses revealed that the differences correlated with amylose-amylopectin content, particle density, compression mechanisms, and TMS between the starch grades. Univariate analysis proved lacking; however, PCA identified the particle size, moisture content, percent crystallinity, amylose-amylopectin ratio, and YP of plastic deformation and elastic recovery as the main factors influencing the starch TMS. SLSR quantified the positive influence of Fourier transform infrared spectra absorbance ratio at 1022-1003 and YP of the immediate elastic recovery, and the negative contribution of amylopectin content on the TMS. Therefore, starch amylose and amylopectin content, crystallinity, and lower elastic recovery are mainly responsible for better TMS.