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Manalis Laboratory - MIT Departments of Biological and Mechanical Engineering

 

Our lab develops and applies novel single-cell measurement approaches with a primary focus on cancer research.  Most projects in the lab stem from two distinct platforms that enable:

1. Ex vivo measurement of single cell biophysical properties
2. In vivo analysis of rare circulating cells in mouse models of cancer

 

1. Ex vivo measurement of single cell biophysical properties
We developed an approach for weighing live single cells with a resolution that is nearly 100-fold better than advanced forms of optical microscopy. The device, known as the suspended microchannel resonator (SMR), is a microfluidic sensor that measures buoyant mass of single cells by detecting a shift in the resonance frequency of a hollow micro-cantilever beam as cells flow through it. With this device, individual mammalian cells can be weighed with a resolution below 100 femtograms which is about 1,000-fold less than the weight of proliferating T cell. This precision has enabled us to study dynamics of cell growth in relation to cell cycle and how cell growth is influenced by perturbations from anti-cancer drugs in ways that have not been possible with existing methods. In addition to weighing cells, the SMR can simultaneously measure cell density as well as deformability. These biophysical measurements can be linked to fluorescence measurements or downstream scRNA-Seq for the same cell. Although our primary focus is on mammalian cells, we have fabricated small channel devices for measuring marine microbes as small as ~10 femtograms.

Future directions: i) While physical properties such as cell volume, density and deformability have been widely studied over the past several decades, buoyant mass (which depends on both the cell’s density and volume) has not. We are currently characterizing buoyant mass of cancer cells and specific immune cell types at defined states with the underlying goal of discovering scenarios where buoyant mass can either be used to identify or enrich for cell types/states that are difficult to find by canonical immunophenotypic markers; ii) Despite tremendous advances in our understanding of cancer pathogenesis, the treatment of individual patients with either conventional chemotherapy or targeted agents remains highly empiric. Pilot studies in GBM, leukemia and multiple myeloma suggest that changes in cancer cell mass following ex vivo treatment can predict patient response. Using cell lines and preclinical models, we are designing experiments aimed at gaining a deeper understanding of the extent to which mass is informative over existing measures of drug response across scales from single cells and clusters to whole organoids; iii) A wide range of process analytical technology (PAT) has been developed for optimizing pharmaceutical manufacturing processes. We are launching new projects to determine the utility of single-cell biophysical properties for various manufacturing processes of adeno-associated viruses.

 

2. In vivo analysis of rare circulating cells in mouse models of cancer
Optofluidic platform for isolating rare cells: Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. To enable these studies of CTC biology in murine cancer models, we developed an optofluidic system capable of detecting and capturing fluorescently labelled cells in awake mice over several hours, days, or weeks. Cannulating a mouse with two permanent catheters, secured on its back, allows for continuous blood withdrawal from the left carotid artery and return through the right jugular vein. Each fluorescent tumor cell that passes through the device emits pulses of light, which allows the controller to open pneumatic valves to redirect a small blood volume that includes the CTC toward a collection tube. Blood from the collection tube can then be purified for CTCs for downstream characterization using techniques such as scRNA-Seq. 

Future directions: Although our work so far has been with CTCs, we are launching new projects where this approach is used to monitor minimal residual disease (MRD) cells in mouse models of hematological cancers and to profile immune cells (e.g. using genetic reporters or in vivo staining).

Measuring kinetics and metastatic propensity of CTCs by blood exchange between mice: Existing pre-clinical methods for acquiring dissemination kinetics of rare CTCs that are en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. We developed an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates between the two mice, we can determine their generation rate and half-life time in circulation.  For small cell lung cancer models, direct transfer of only ~1% of daily-shed CTCs using our blood-exchange technique from late-stage tumor bearing mice can generate macrometastases in healthy recipient mice.

Future directions: Our blood-exchange technique can provide accurate identification of the rate-limiting steps in the blood transport phases of the metastatic cascade. We are currently using it within two on-going efforts in the lab: i) studying tumor progression by generating macrometastases in healthy recipient mice from tumor bearing mice at various stages of solid tumor development, as well at MRD stages of hematological tumors; ii) directly and controllably exchanging other blood components in order to study trafficking dynamics of immune cells in various biological contexts within immunology, cancer biology, and aging. Because our blood-exchange technique can be used continuously and longitudinally, it can potentially reveal temporal kinetics that occur on the order of minutes, hours, or days and hence assist in establishing suitable time windows for maximizing therapeutic efficacy.

 

 

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