Statins abrogate gemcitabine-induced PD-L1 expression in pancreatic cancer-associated fibroblasts and cancer cells with improved therapeutic outcome.

A combination of chemotherapy with immunotherapy has been proposed to have better clinical outcomes in Pancreatic Ductal Adenocarcinoma (PDAC). On the other hand, chemotherapeutics is known to have certain unwanted effects on the tumor microenvironment that may mask the expected beneficial effects of immunotherapy. Here, we have investigated the effect of gemcitabine (GEM), on two immune checkpoint proteins (PD-L1 and PD-L2) expression in cancer associated fibroblasts (CAFs) and pancreatic cancer cells (PCCs). Findings of in vitro studies conducted by using in-culture activated mouse pancreatic stellate cells (mPSCs) and human PDAC patients derived CAFs demonstrated that GEM significantly induces PD-L1 and PD-L2 expression in these cells. Moreover, GEM induced phosphorylation of STAT1 and production of multiple known PD-L1-inducing secretory proteins including IFN-γ in CAFs. Upregulation of PD-L1 in PSCs/CAFs upon GEM treatment caused T cell inactivation and apoptosis in vitro. Importantly, Statins suppressed GEM-induced PD-L1 expression both in CAFs and PCCs while abrogating the inactivation of T-cells caused by GEM-treated PSCs/CAFs. Finally, in an immunocompetent syngeneic orthotopic mouse pancreatic tumor model, simvastatin and GEM combination therapy significantly reduced intra-tumor PD-L1 expression and noticeably reduced the overall tumor burden and metastasis incidence. Together, the findings of this study have provided experimental evidence that illustrates potential unwanted side effects of GEM that could hamper the effectiveness of this drug as mono and/or combination therapy. At the same time the findings also suggest use of statins along with GEM will help in overcoming these shortcomings and warrant further clinical investigation.

Therapeutic role of N-acetyl cysteine (NAC) for the treatment and/or management of SARS-CoV-2-induced lung damage in hamster model.

Oxidative stress by reactive oxygen species (ROS) has been hypothesized to be the major mediator of SARS-CoV-2-induced pathogenesis. During infection, the redox homeostasis of cells is altered as a consequence of virus-induced cellular stress and inflammation. In such scenario, high levels of ROS bring about the production of pro-inflammatory molecules like IL-6, IL-1ß, etc. that are believed to be the mediators of severe COVID-19 pathology. Based on the known antioxidant, anti-inflammatory, mucolytic and antiviral properties of NAC, it has been hypothesized that NAC will have beneficial effects in COVID-19 patients. In the current study efforts have been made to evaluate the protective effect of NAC in combination with remdesivir against SARS-CoV-2 induced lung damage in the hamster model. The SARS-CoV-2 infected animals were administered with high (500 mg/kg/day) and low (150 mg/kg/day) doses of NAC intraperitoneally with and without remdesivir. Lung viral load, pathology score and expression of inflammatory molecules were checked by using standard techniques. The findings of this study show that high doses of NAC alone can significantly suppress the SARS-CoV-2 mediated severe lung damage (2 fold), but on the contrary, it fails to restrict viral load. Moreover, high doses of NAC with and without remdesivir significantly suppressed the expression of pro-inflammatory genes including IL-6 (4.16 fold), IL-1ß (1.96 fold), and TNF-α (5.55 fold) in lung tissues. Together, results of this study may guide future preclinical and clinical attempts to evaluate the efficacy of different doses and routes of NAC administration with or without other drugs against SARS-CoV-2 infection.

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-ß-induced tumor-associated fibrosis.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-ß-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.