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Reduced CTL motility and activity in avascular tumor areas

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Abstract

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.

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Abbreviations

ALP-red:

Alkaline phosphatase red

CFP:

Cyan fluorescent protein

DAB:

3,3′-Diaminobenzidine

DIC:

Differential interference contrast

FAS:

First apoptosis signal

FasL:

Fas ligand

FCCP:

Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone

HIF-1α:

Hypoxia inducible factor 1 α

IRES:

Internal ribosome entry site

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Funding

Guy Shakhar and Tali Feferman were supported in this project by grants from the Israel Science Foundation (Grant 1735/15), the Israel Cancer Association (Grant no. 20170182), and from the German Cancer Research Center and the Israeli ministry of Science and Technology (DKFZ-MOST) (Grant no. GR2353) collaborative program.

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Authors and Affiliations

  1. Department of Immunology, Weizmann Institute of Science, Wolfson Bldg., 234 Herzl St., 76100, Rehovot, Israel

    Yoav Manaster, Zohar Shipony, Anat Hutzler, Masha Kolesnikov, Tali Feferman & Guy Shakhar

  2. Department of Pathology, Sheba Medical Center, Derech Sheba 2, 52621, Ramat Gan, Israel

    Camila Avivi, Bruria Shalmon & Iris Barshack

  3. Ella Institute, Sheba Medical Center, Derech Sheba 2, 52621, Ramat Gan, Israel

    Michal J. Besser

  4. Department of Human Microbiology and Immunology, Sackler Medical School, Tel-Aviv University, 35 Klachkin st, 6997801, Tel Aviv, Israel

    Michal J. Besser

Authors
  1. Yoav Manaster
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  2. Zohar Shipony
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Contributions

YM and TF performed the experiments and wrote the paper, AH and GS performed additional experiments, ZS assisted with analysis, and MK assisted with data visualization. CA and BS prepared clinical samples, IB and MJB provided these samples, and GS conceived and planned the project and helped writing the paper.

Corresponding authors

Correspondence to Tali Feferman or Guy Shakhar.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

Experiments were approved by the institutional animal care and use committee and the Institutional Review Board at the Weizmann Institute of Science. Protocol number 40351217-2. We followed the guidelines for animal use in research in Israel and for the use of specimens of human origin.

Informed consent

All patients signed a form consenting to the use of diagnostic samples taken from them for anonymous, non-commercial research purposes.

Animal source

C57BL/6 mice were purchased from Harlan laboratories Ltd. All other mouse strains were purchased from the Jackson Laboratories and bred locally.

Cell line authentication

Cells from the B16a melanoma line [25] were kindly provided by Prof. Bonnie Sloane at Wayne State University, Detroit, MI. Cells were periodically tested for the presence of mycoplasma and found clean. All lines were used within ten passages. Expression of OVA and fluorescent proteins after cell passages was confirmed by flow cytometry. All tumor lines grew rapidly in untreated immunocompetent syngeneic C57BL/6 mice and formed pigmented tumors typical of melanoma.

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Manaster, Y., Shipony, Z., Hutzler, A. et al. Reduced CTL motility and activity in avascular tumor areas. Cancer Immunol Immunother 68, 1287–1301 (2019). https://doi.org/10.1007/s00262-019-02361-5

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