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Singh Lab Research Areas

 

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Singh Laboratory: Immunotherapy and Cell Engineering Laboratory

 

IMMUNO-ENGINEERING

Engineered Immune Organoids and Lymphatics-on-chip

We are interested in developing ex vivo models of the immune system that recapitulate zones of lymph nodes and lymphatics. In one of the key innovations, we have developed an ex vivo B cell follicle, a three-dimensional tissue that is capable of undergoing germinal center reaction. In another innovation, we have developed on-chip microfluidic devices that recapitulate lymphatic fluid charactistics and demonstrate their effect on tumors of lymph node. 

B cell follicle and germinal center organoids: Humoral immunity against infections depends on the germinal center (GC) reaction occurring in the B cell follicles of lymphoid tissues, e.g. lymph nodes.  Upon T cell dependent activation, naïve B cells form GCs, wherein they undergo massive proliferation and somatic hypermutation of their immunoglobulin loci, to eventually form antibody secreting plasma cells. The process of GC reaction is further controlled by epigenetic switches. A major impediment to detailed mechanistic studies of normal and transformed GC B cells, as well as the ability to generate high affinity, antibody secreting cells in vivo, is the lack of experimental systems that sufficiently recapitulate the biology of this complex biological system. Overcoming this limitation requires the ability to manipulate and generate such cells in a tractable system. Our work has led to the development of a first immune tissue in a dish that recapitualtes selective aspect of lymph nodes and germinal centers. See our Nature Communications, Nature Protocols, Biomaterials, and ACS Biomaterials Science and Engineering papers. Our interest areas include:

(1) Understanding of factors that control B and T cell immune response against threats;

(2) Rapid development and translation of large number of activated B cells capable of producing disease-specific antibodies;

(3) Rational development and evaluation of immunotherapeutics and vaccines against HIV, Zika, and other infectious diseases, and

(4) Understanding of matrix immunology and B cell receptor signaling

This research was highlighted as Top 100 discoveries of 2015 by the Discover Magazine and a recipient of 2015 Biomaterials Outstanding Paper Award from Elsevier. 

See Cornell Chronicle: http://news.cornell.edu/stories/2015/06/engineers-synthetic-immune-organ-produces-antibodies

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Figure: Schematic with cellulat, biophysical, and biochemical components of lymph node (Panel A). Ex vivo 3D bioengineered immune organoids induce rapid differentiation of naive B cells into germinal center-like phenotype within 4 days and at ~ 100-fold faster rate than conventional ex vivo immunology approaches (Panel B). The whole transcriptome is comparable to immunized mice (Panel C) and enabled a recent discovery of epigenetic modulator of germinal center process (Panel D; Nature Communications, 2017)

Lymphatic-on-Chip like devices to model fluid force effect on immunity and tumors: The immune cells and tumors that originate in lymph nodes are exposed to lymphatic fluid forces. In a recent study published in Cell Reports, we have developed a lymphatic-on-chip like device that recapitulates flow inside lymph vessels and parts of lymph node. We used this technology to describe the role of fluid forces, from lymphatics and neo-vessels, in mechanomodulation of integrin and B cell receptor signaling. These insights shed light on the heterogeneous nature of lymphomas and may allow faster translation of therapeutics. This is the team’s first step toward modeling lymphatic systems and the effects of fluid flow on them, which we plan to further modify with relevant lymphatic cells and use for understanding immunity and malignancy.

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Apoorva et al. Cell Reports, 2018, 23(2), 499-511

Access paper here: http://www.cell.com/cell-reports/pdf/S2211-1247(18)30430-3.pdf

Hydrogel-based 3D Lymphoma Tumor Microenvironment

Tumor growth and survival are maintained by cellular and molecular mechanisms linked either to abberant mutations or to normal proteins hijacked by tumors  to their advantaage. Targeting hallmark proteins and pathways holds the promise to improve efficacy and decrease toxicity of anticancer treatments. However, even targeted therapies are unlikely to be curative unless their therapeutic efficacy is evaluated under appropriate tumor microenvironemnt that represent complexity, heterogeneity, feedback mechanisms, and propensity to develop resistance. Our research focus has been on understanding the role of lymphoid tumor microenvironment in B and T cell lymphomas and developing innovating biomaterials-based platform technologies to determine tumor heterogeneity and causes of  drug resistance in lymphoma patients. We apply concepts of tissue mechanics, tissue bio-adhesivity, and fluid mechanics integrated with cancer cell biology to engineer platform technologies that enable fundamental discoveries in cancers biology and rational translation of therapeutics. Our recent findings with collaborators at Weill Cornell Medical College, published in Blood, demonstrated the role of integrin signaling in patient-derived T cell lymphoma survival and progression, in vitro and vivo.

In a parallel study published in Biomaterials, we have provided evidence that integrins are critical for the growth, clustering, BCR activity, and chemo-resistance of activated B cell-like Diffuse Large B cell lymphoma (ABC-DLBCL), which are the most chemo-resistant lymphomas (5-year overall survival ~ 30%). These biological discoveries led to the development of the first 3D lymphoma organoids (hydrogels) that presented lymphoma-specific integrin ligands to ABC-DLBCL and induced enhanced proliferation, cell signaling, and drug resistant. 

See Cornell Chronicle: http://www.news.cornell.edu/stories/2015/10/3-d-organoids-allow-tests-lymphoma-treatments

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Figure: B cell lymphomas grow in bulk and micro organoids as clusters, similar to those in patients. The green fluorescent areas represent lymphoma cells
 

Nanotechnologies for modulating immune cells for cancer immunotherapy

Current immunotherapy approaches often result in partial immunity that predisposes the patient to a risk of reinfection or serious toxicity issues. In Singh Lab, we focus on developing nano-engineered technologies to modulate immune cells and program them for cancer immunotherapy.

To that end, we have engineered a self-assembly protein-biomaterial nanogel system for inducing robust immunity at low protein doses. Our more recently funded project with Prof. Brian Rudd at Cornell Immunology focuses on developing nanoneedles for cancer immunotherapy.

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