Background Intricate nature within tumors presents major obstacle in successful cancer management. A self-renewing subset of stem cells, designated as cancer stem cells (CSCs), drives this heterogeneity. Truncation of effector response from infiltrated CD8+ T-cells enhance Breast cancer stem cells (BCSC) survival.^1–3 Within tumor microenvironment (TME), CD8⁺ T-cells undergo a hierarchical loss of proliferation and effector functions. However, the crosstalk between BCSCs and CD8⁺ T-cells are poorly understood.

2-deoxyglucose (2DG), a glucose analogue used as a metabolic regulator, inhibits glycolysis and augments CD8⁺ T-cell infiltration into the TME, potentially enhancing their cytotoxic activity. Given the eminence of BCSCs in metastatic cancer progression by evading therapies, we became interested to study the possible crosstalk between CD8⁺ T-cells and BCSCs in tumor advancement within BC sub-types and intervention of 2DG in regulating the same.

Methods MACS-sorted BCSCs (Lin^-CD44⁺CD24^-) and T-cells (Lin⁺CD8⁺T-cell) were isolated from patients’ tumor. These CD8⁺T cells were co-cultured with BC sub-types to study the interaction with CD8⁺T-cells. Further the influence of 2DG was elucidated. Flow-cytometry, Western-blot, RT-PCR were performed to identify involved transcription factors (KLRG1, Granzyme B, Tox, Tim3, PD1 etc.), stem-cell markers (CD44⁺ CD24^-) and CD8⁺T-cell (PD1, KLRG1, IFNγ, GrB) within BC.

Results A positive co-relation between BCSCs and CD8⁺PD1^highKLRG1^highIFNγ^low T-cells was noted in TNBC cells compared to other subtypes. Exhausted CD8⁺T-cells (PD1^highTOX⁺Eomes⁺CXCR5⁺) collaborated with elevated BCSC level. Exposure of TNBC-BCSCs in T-cell co-culture suggested a prominent gain of exhaustion features with a significant loss of effector function within CD8+T-cells. Addition of 2DG partially retrieving CD8 ⁺ T-cell from their dysfunction by lowering the status of KLRG1 and PD-1. Simultaneously, 2DG helps better CD8⁺ T- cell mediated killing by enhancing IFNγ, Granzyme-B secretion. 2DG might restrict BCSCs while boosting CD8 ⁺ T-cells. Corrupted CD8⁺ T-cells significantly enhance the tumorigenic potential of BCSCs. The modulation of BCSCs and T-cells by 2DG and its potential implications in breast cancer management was also discerned (figure 1).

Conclusions Study discloses strong association between exhausted CD8⁺ T-cell population and CSCs in BC, particularly in TNBC indicating their possible association in tumor promotion. The exhaustion status of CD8+ T-cells might be influenced by BCSCs and overall immune infiltration of the TME. This aligns with the increase in BCSCs and corresponded to poor prognosis. 2DG alleviates dysfunctional-CD8⁺T cell by interfering BCSC and T-cell crosstalk. Therefore, 2DG could be utilized as prospective therapeutic targets against CD8⁺T-influenced aggressive BCSCs and it provides a new nontoxic therapeutic alternative to target BCSCs.

Acknowledgements We thank Director, Chittaranjan National Cancer Institute, Kolkata, India, for providing institutional facilities. Special thanks to Dr. Niyaz Alam, for providing patient sample, CNCI, Kolkata. We also thank all members of our respective laboratories for their technical support for this work.

References

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2. Sasaki K, Nishino S, Yamaguchi A, Fukuda K, Hard Y, Yamamura M, Egashira K, Rhino K. Nanoparticle-mediated’ delivery of 2-deoxy-D-glucose induces anti tumor immunity cytotoxicity in liver tumors in mice. Cellular Molecular Gastroenterology Hepatology 2021 Jan 1;11(3):739–62.

3. Guha A, Goswami KK, Sultana J, Ganguly N, Choudhury PR, Chakravarti M, Bhuiyan A, Sarkar A, Bera S, Dhar ‘S,’ Das J. Cancer stem cell–immune cell cross talk in breast tumor micro environment: determinant of therapeutic facet. Frontiers in Immunology 2023 Nov 27;14:1245421.

Ethics Approval All human experiments were approved by Institutional Human Ethical Committee of Chittaranjan National Cancer Institute, Kolkata, India. Approval numbers are CNCI-IEC-SB-20. All patients included gave informed consent before taking part in the study.

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Abstract 930 Figure 1

Model proposing BCSC and CD8+T cell crosstalk