A multiplexed microfluidic system for evaluation of dynamics of immune-tumor interactions.

Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.

Macrophages and pancreatic islet amyloidosis.

Islet amyloid formed from islet amyloid polypeptide (IAPP, amylin) is found in spontaneously diabetic monkeys and cats. Islet amyloidosis is progressive, apparently irreversible and is associated with destruction of insulin-secreting cells. The role of macrophages in the destruction and removal of islet amyloid is unknown. Therefore, the presence and morphology of macrophages were determined by electron and quantitative light microscopy in islets of diabetic and nondiabetic man and monkeys and in transgenic mice expressing the gene for human IAPP. Tissue macrophages were present in all pancreatic sections and tissue distribution was similar in exocrine and endocrine areas. There was no difference in macrophage density in amyloidotic and amyloid-free islets in monkeys and man. Macrophage density was similar in islets of transgenic mice expressing human IAPP which do not contain amyloid in vivo but in which fibrils are formed in vitro following islet isolation compared to islets from mice expressing rat IAPP which is not amyloidogenic. IAPP amyloid fibrils were visible by electron microscopy in lysosomes of pancreatic macrophages in man, monkeys and human IAPP transgenic mice. Thus, human IAPP is internalised but inefficiently degraded by tissue macrophages. Diabetes-associated amyloidosis is not associated with visible recruitment of macrophages for removal of amyloid or islet debris.

Relationship between the maximum velocity and sodium affinity of the erythrocyte sodium pump and its rate constant in blood.

1. Young and mature erythrocytes from 15 normal subjects were used to compare the sodium pump rate constant measured in whole blood with the more definitive sodium affinity constant and maximum velocity of the sodium pump measured in artificial media using sodium-loaded cells. 2. Similar values were obtained from both erythrocyte fractions for the sodium affinity constant and maximum velocity and also by using two different plots. The median error in the estimate of individual sodium affinity constants and maximum velocities from regression analysis was about 20% and the precision was not improved by combining the data points for the two erythrocyte fractions. 3. The rate constant in whole blood was closely related to the sodium affinity constant and maximum velocity of the sodium pump (r = 0.75), suggesting that it was a reasonable overall assessment of available sodium pump activity. 4. Differences in the rate constant between subjects were due to differences in both the maximum velocity and sodium affinity constant of the sodium pump so that the rate constant could not be used as a guide to the underlying sodium pump physiology.

The renin-angiotensin-aldosterone system in decompensated cirrhosis: its activity in relation to sodium balance.

Plasma renin activity (PRA), plasma renin concentration (PRC), plasma angiotensin II concentration (AII), plasma and urinary aldosterone (PA, UA) and urinary sodium excretion (UNaV) were measured in 51 normal controls, 16 patients with decompensated cirrhosis (i.e. ascites and/or oedema present) in sodium equilibrium (Group 1) and 13 patients with decompensated cirrhosis in a phase of active sodium retention (Group 2). In Group 1 the mean supine and erect values, although lower, were not significantly different from controls. In Group 2 the mean values were significantly elevated, but several individual values were within the normal range; there were significant direct relationships between plasma renin activity and plasma renin concentration (r = 0.85, p less than 0.001 erect), plasma renin concentration and plasma angiotensin II concentration (r = 0.86, p less than 0.001 erect), and plasma angiotensin II concentration and plasma aldosterone (r = 0.70, p less than 0.01 erect). In Group 2 there was an inverse correlation between urinary sodium excretion and both urinary aldosterone (r = -0.50) and erect plasma aldosterone (r = -0.36) but, perhaps because of the narrow range of sodium excretion rates, significance was not reached. The normal values in Group 1 indicate that hyperaldosteronism is not essential for the maintenance of established ascites, but do not exclude a role for aldosterone in the control of sodium excretion if it is accepted that renal tubular sensitivity to aldosterone is increased in these patients. In Group 2, the raised mean plasma and urinary aldosterone levels and the trend towards an inverse relationship with urinary sodium excretion suggests a role for aldosterone in the active retention of sodium. It appears that stimulation of the renin-angiotensin system is the major factor in the elevation of plasma aldosterone; there was no relationship between plasma aldosterone and either plasma sodium or potassium levels. The mechanism of renin hypersecretion is unclear but this may represent part of a sympathetically mediated response in order to maintain blood pressure. The close relationship between plasma renin activity and plasma renin concentration indicates that the former is a valid measure of circulating renin levels in cirrhosis, despite low renin-substrate levels.