Nuclear and nuclear envelope remodelling during melanoma metastasis and immune control

Primary Supervisor: Jeremy Carlton, King’s College London

Secondary supervisor: Victoria Sanz Moreno, Queen Mary University of London

Project

Cancer cells, stromal cells and the extracellular matrix form a complex tumour microenvironment (TME). Cancer cells must squeeze through constraints in this TME during metastatic dissemination and during the host anti-tumour response, immune cells must infiltrate this complex tissue to target cancer cells. As the largest and stiffest organelle in the cell, the nucleus represents the most significant barrier to constrained migration. We want here to understand how cancer cells and immune cells remodel this organelle to enable their efficient migration. We have recently discovered a role for an inner nuclear membrane protein called LAP1 in allowing remodelling of the nuclear envelope to permit cancer constrained migration, enabling metastatic spread of melanoma cells. In this project, we have three main aims.

1. To understand at the ultrastructural level how the nucleus of melanoma cells is remodelled to facilitate migration in 3D environments
2. To explore whether immune cells require nuclei and nuclear envelope remodelling to enable tumour infiltration, and to understand what role LAP1 plays in T cell migration.
3. To develop 3D-co-culture assays to simultaneously track cancer cell migration, genetically modified T cell migration and T-cell-mediated killing of melanoma cells.

Firstly, we will use correlative 3D-electron microscopy to explore how the nucleus of melanoma cells is shaped during migration in collagen matrices and in tissues. Working with the CRICK EM-core, we will perform a world first in the volumetric EM-imaging and reconstruction of migrating melanoma cells in 3D-collagen. We will also process tissue from human and mouse melanomas undergoing local dermal invasion to explore this process at the ultrastructural level. We will use machine learning to reconstruct nuclei and extract prognostic information from these datasets. Building on our published data, we will examine how the inner nuclear membrane protein, LAP1, controls nuclear shape and influences constrained cancer cell migration.

Secondly, we will explore how immune cells migrate into tumours and we will understand whether they are similarly restricted by the TME constraints and the need for nuclear remodelling. Excitingly, we have discovered that LAP1 is highly expressed in professional migratory immune cells, notably CD8 T cells and macrophages. We have developed digital pathology approaches to document LAP1 expression and will use them here to assess LAP1 expression in the immune infiltrate of mouse and human tumours.

Finally, we will generate LAP1 knockout T-cells and explore their ability to migrate into melanomas. We will examine nuclear and nuclear envelope remodelling in this cells and apply our understanding of LAP1 biology to rescue T-cell migration with LAP1 mutants. We will also edit the TCR locus of CD8+ T-cells to allow them to target melanoma cancer cells, allowing us to assess melanoma killing in-vivo and in-vitro and to explore whether nuclear rearrangements are rate-limiting for tumour infiltration and killing.

Candidate background

This PhD would suit an enthusiastic candidate with experience in microscopy and biochemistry and molecular cell biology. We can provide training in electron microscopy, image reconstruction, histology, cell migration, genome editing and in vivo mouse work.

Potential Research Placements

1. Electron Microscopy Science Technology Platform, Francis Crick Institute
2. James Lee, Francis Crick Institute
3. Frances Balkwill, Barts Cancer Institute/ Queen Mary University of London

Reference

1. Jung-Garcia Y, Maiques O, Rodriguez-Hernandez I, Fanshawe B, Domart MC, Renshaw M, Marti RS, Matias-Guiu X, Collinson LM, Sanz-Moreno V* & Carlton JG* (2021) LAP1 regulates nuclear plasticity to enable constrained migration. bioRxiv doi: 10.1101/2021.06.23.449503 *co-corresponding co-senior authors.
2. Orgaz JL, Crosas-Molist E, Sadok A, Perdrix-Rosell A, Maiques O, Monger J, Rodriguez-Hernandez I, Mele S, Georgouli M, Bridgeman V, Karagiannis P, Pandya P, Cantelli G, Boehme L, Wallberg F, V Tape C, Karagiannis SN, Malanchi I, Sanz-Moreno V (2020) Myosin II reactivation and Cytoskeletal remodelling as a hallmark and a vulnerability in melanoma resistance. Cancer Cell, 37, 85-103. 10.1016/j.ccell.2019.12.003
3. Georgouli M, Herraiz C, Crosas-Molist E, Fanshawe B, Maiques O, Perdrix A, Pandya P, Cantelli G, Rodriguez-Hernandez I, Karagiannis P, Lam H, Josephs D, Matias-Guiu X, Marti RM, Nestle FO, Orgaz JL, Malanchi I, Fruhwirth GO, Karagiannis SN and Sanz-Moreno V (2019) Regional activation of Myosin II in cancercells drives tumour progression via a secretory cross-talk with the immune microenvironment. Cell. 176:757-774. doi: 10.1016/j.cell.2018.12.038
4. Gatta AT, Olmos Y, Stoten CL, Chen Q, Rosenthal PB and Carlton JG* (2021). CDK1 controls CHMP7-dependent nuclear envelope reformation. eLife 10:e59999 doi: 10.7554/eLife.59999
5. Olmos Y, Hodgson L, Mantell J, Verkade P and Carlton JG* (2015). ESCRT-III controls nuclear envelope reformation. Nature 522, 236-39 doi: 10.1038/nature14503