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An unbiased in vivo functional genomics screening approach in mice identifies novel tumor cell-based regulators of immune rejection

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Abstract

The clinical successes of immune checkpoint therapies for cancer make it important to identify mechanisms of resistance to anti-tumor immune responses. Numerous resistance mechanisms have been identified employing studies of single genes or pathways, thereby parsing the tumor microenvironment complexity into tractable pieces. However, this limits the potential for novel gene discovery to in vivo immune attack. To address this challenge, we developed an unbiased in vivo genome-wide RNAi screening platform that leverages host immune selection in strains of immune-competent and immunodeficient mice to select for tumor cell-based genes that regulate in vivo sensitivity to immune attack. Utilizing this approach in a syngeneic triple-negative breast cancer (TNBC) model, we identified 709 genes that selectively regulated adaptive anti-tumor immunity and focused on five genes (CD47, TGFβ1, Sgpl1, Tex9 and Pex14) with the greatest impact. We validated the mechanisms that underlie the immune-related effects of expression of these genes in different TNBC lines, as well as tandem synergistic interactions. Furthermore, we demonstrate the impact of different genes with previously unknown immune functions (Tex9 and Pex14) on anti-tumor immunity. Thus, this innovative approach has utility in identifying unknown tumor-specific regulators of immune recognition in multiple settings to reveal novel targets for future immunotherapies.

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Abbreviations

BCA:

Bicinchoninic acid assay

CaeCam2:

Carcinoembryonic antigen related cell adhesion molecule 2

CCK:

Cholecystokinin

CCL4:

C-C motif chemokine ligand 4

CO2:

Carbon dioxide

CRISPR:

Clustered regularly interspaced short palindromic repeats

ENPP2:

Ectonucleotide pyrophosphatase/phosphodiesterase 2

FDR:

False discovery rate

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HCL:

Hydrogen chloride

IDO:

Indoleamine-pyrrole 2,3 dioxygenase

IgG:

Immunoglobulin G

IL-4:

Interleukin 4

IL13Ra1:

Interleukin 13 receptor subunit alpha 1

KD:

Knocked down

LIMMA:

Linear models for microarray data

MOI:

Multiplicity of infection

NSG:

NOD-SCID-IL2 gamma chain knock-out

NaCl:

Sodium chloride

OE:

Overexpression

OX40L:

Tumor necrosis factor (ligand) superfamily, member 4

P53:

Tumor protein P53

Pex14:

Peroxisomal membrane protein 14

RNAi:

RNA interference

S1P:

Sphingosine-1-phosphate

SBI:

System biosciences

SEMA7a:

Semaphorin 7a

Sgpl1:

Sphingosine-1-phosphate ligase 1

shRNA:

Short harpin RNA

SMAD3:

SMAD family member 3

Spns2:

S1P transporter spinster homologue 2

Tex9:

Testis expressed gene 9

Tiam1:

T-Cell lymphoma invasion and metastasis 1

TME:

Tumor microenvironment

TNBC:

Triple-negative breast cancer

TSP1:

Thrombospondin 1

WT:

Wild-type

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Acknowledgements

This research was supported by the following Shared Resources at Lombardi Comprehensive Cancer Center: The Genomics and Epigenomics Shared Resource, the Flow Cytometry and Cell Sorting Shared Resource, and the Tissue Culture Shared Resource. All Lombardi Comprehensive Cancer Center Shared Resources are partially supported by National Institutes of Health (NIH)/National Cancer Institute (NCI) Grant P30-CA051008. An antibody to CD47 was kindly provided by Dr. Robert Karr, Tioma Therapeutics, Inc (St. Louis, MO).

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Authors and Affiliations

  1. Department of Oncology and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington DC, 20057, USA

    Casey W. Shuptrine, Reham Ajina, Sandra A. Jablonski & Louis M. Weiner

  2. Department of Oncology, Division of Biostatistics, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA

    Elana J. Fertig

  3. Department of Surgery, Duke University Medical Center, Durham, NC, USA

    Casey W. Shuptrine, H. Kim Lyerly & Zachary C. Hartman

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  1. Casey W. Shuptrine
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  2. Reham Ajina
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  3. Elana J. Fertig
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  4. Sandra A. Jablonski
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  5. H. Kim Lyerly
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  6. Zachary C. Hartman
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  7. Louis M. Weiner
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Correspondence to Louis M. Weiner.

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This manuscript was supported by NIH Grants CA50633 (Louis M Weiner) and CA51880 (Louis M Weiner), and Susan G. Komen Career Catalyst Research Grant CCR14299200 (Zachary C Hartman).

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The authors have no conflicts of interest to report or disclose.

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Shuptrine, C.W., Ajina, R., Fertig, E.J. et al. An unbiased in vivo functional genomics screening approach in mice identifies novel tumor cell-based regulators of immune rejection. Cancer Immunol Immunother 66, 1529–1544 (2017). https://doi.org/10.1007/s00262-017-2047-2

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