Background Poor infiltration of therapeutic cells to solid tumors is a major hurdle to success of cell-based therapies.¹Upon intravenous administration, only a small fraction (<2%) of these cells infiltrate the tumor.^{2 3} One of the mechanisms driving exclusion of immune cells from solid tumors is the endothelial cell anergy of angiogenic vessels. Angiogenic vessels have significantly lowered leukocyte rolling and cell adhesion.^{4 5} Engineering approaches exploiting the altered phenotype of angiogenic vessels to promote cell adhesion could increase tumor infiltration. We hypothesized that the molecular cues left behind by disseminating cancer cells could provide a molecular track that could be traced by therapeutic T-cells. To test this hypothesis, we take advantage of Biglycan, a secreted proteoglycan, that is frequently overexpressed on tumor endothelial cells (TECs) in metastatic tumors.^{6 7} To enable T-cells to bind to biglycan, we drew inspiration from bacteria of genus Borrelia which disseminate to different host organs in lyme disease by decorin binding proteins A and B (DbpA/B) mediated binding to biglycan on endothelial cells.⁸ We hypothesized that heterologous expression of DbpA/B on T-cells would enable their rolling and adhesion on angiogenic vessels.

Methods We used bulk and spatial transcriptomic datasets on human cancers to investigate expression of biglycan in metastatic cancers. We cloned plasmid constructs for expressing Borrelia garinii DbpA/B fused to different signal sequences to heterologously express these proteins on human T-cells (hereafter Sticky T-cells). We used flow-cytometry, fluorescent microscopy and microfluidic flow-based assays to quantify adhesion of Sticky T-cells flowing over biglycan coated surfaces and human umbilical vein endothelial cells (HUVECs).

Results Biglycan (BGN) is highly expressed in metastatic ovarian, skin, stomach and kidney tumors. The expression of BGN is strongly correlated with angiogenesis markers.

Spatial transcriptomic analysis of human ovarian and lung cancers shows BGN overexpression on TECs and T-cell exclusion in BGN rich regions.

Sticky T-cells overexpressing DbpA/B fused to a type II single pass membrane signal sequence in T cells enables their binding to biglycan.

Sticky T-cells roll more frequently and with lower velocities than control T-cells over biglycan coated surfaces and HUVECs.

Sticky T-cells infiltrate xenotransplant models (melanoma and ovarian) at higher frequencies compared to unmodified T-cells.

Conclusions Our findings indicate that Sticky T-cells efficiently infiltrate metastatic solid tumors. This strategy holds potential in overcoming a major barrier to the efficient trafficking of these cells to the tumor.

References

1. Albelda SM. CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn. Nat Rev Clin Oncol 2024;21:47–66. 10.1038/s41571-023-00832-4.

2. Chapelin F, Gao S, Okada H, Weber TG, Messer, K, Ahrens ET. Fluorine-19 nuclear magnetic resonance of chimeric antigen receptor T cell biodistribution in murine cancer model. Sci Rep 2017;7:17748. 10.1038/s41598-017-17669-4.

3. Skovgard MS, Hocine HR, Saini JK, Moroz M, Bellis RY, Banerjee S, Morello A, Ponomarev V, Villena-Vargas J, Adusumilli PS. Imaging CAR T-cell kinetics in solid tumors: translational implications. Mol Ther Oncolytics 2021;22:355–367. 10.1016/j.omto.2021.06.006.

4. Jain RK, Koenig GC, Dellian M, Fukumura D, Munn LL, Melder RJ. Leukocyte-endothelial adhesion and angiogenesis in tumors. Cancer Metastasis Rev 1996;15:195–204. 10.1007/BF00437472.

5. Dirkx AE, Oude Egbrink MG, Kuijpers MJ, van der Niet ST, Heijnen VV, Bouma-ter Steege JC, Wagstaff J, Griffioen AW. Tumor angiogenesis modulates leukocyte-vessel wall interactions in vivo by reducing endothelial adhesion molecule expression. Cancer Res 2003;63:2322–2329.

6. Yamamoto K, Ohga N, Hida Y, Maishi N, Kawamoto T, Kitayama K, Akiyama K, Osawa T, Kondoh M, Matsuda K, et al. Biglycan is a specific marker and an autocrine angiogenic factor of tumour endothelial cells. Br J Cancer 2012;106:1214–1223. 10.1038/bjc.2012.59.

7. Maishi N, Ohba Y, Akiyama K, Ohga N, Hamada J, Nagao-Kitamoto H, Alam MT, Yamamoto K, Kawamoto T, Inoue N, et al. Tumour endothelial cells in high metastatic tumours promote metastasis via epigenetic dysregulation of biglycan. Sci Rep 2016;6:28039. 10.1038/srep28039.

8. Salo J, Pietikäinen A, Söderström M, Auvinen K, Salmi M, Ebady R, Moriarty TJ, Viljanen MK, Hytönen J. Flow-tolerant adhesion of a bacterial pathogen to human endothelial cells through interaction with biglycan. J Infect Dis 2016;213:1623–1631. 10.1093/infdis/jiw003.