1
SM Kirov Military Medical Academy, Saint Petersburg, Russian Federation
2
II Mechnikov North-West State Medical University, Saint Petersburg, Russian Federation
3
Alternative Innovative Technologies, LLC, Boston, MA, United States
Corresponding author details:
Kravtsov V Yu
SM. Kirov Military Medical Academy
Saint Petersburg
Russian Federation
Copyright:
© 2020 Chuchalin EO, et al.
This is an open-access article distributed
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Attribution 4.0 international License, which
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We studied the interaction of PD-1+ lymphocytes with Ovary Carcinoma Cancer Cells
(OCC) in several patients with pleural metastasis using immunocytochemical staining
of pleural liquid. In addition to the intact separate T-cells, we discovered ubiquitous PD1+
-OCC aggregates in which the lymphocytes showed significant signs of cell defects and
degradation in the environment of the otherwise intact OCC. Formation of such complexes
and subsequent destruction of lymphocytes may be a pathway to provide the nutritional
supply and support further growth of cancer cells in plank tonic state, in absence of
vascularization and angiogenesis, relevant to both progression of lung metastases and
some primary tumors such as small cell lung carcinoma.
Ovarian Carcinoma; T-Lymphocytes; Pleural Effusion
High aggression of the metastatic tumors, particularly ubiquitous in the lungs, remains one of the major problems in managing cancer progression and treatment. For example, the pleural metastatic expression of Ki-67 is high whereas p53 is lower than in primary tumors [1]. However, an establishment and growth of early metastatic formations are still poorly understood and not really amenable to prevention. For the most part the metastatic tumor proliferation is considered in context of interaction with the related stromal cells, vascularization and angiogenesis.
Tumor Infiltrating Lymphocytes (TIL) are promising agents in cancer therapy as
indicated by ongoing clinical trials involving autologous isolates causing tumor lysis [2].
Development of the PD-1+
sub-population of activated lymphocytes has been linked to
cancer progression and various drug strategies emerged related to the so-called immune
checkpoint inhibitors (e.g., Keytruda) blocking this receptor. In this report, driven by the
interest in metastatic tumors and the role of T-cells, we looked into interactions of plank
tonic cancer cells with circulating lymphocytes in pleural isolates of several patients with
metastatic ovary carcinoma and discovered widespread PD-1+
-OCC aggregates that are
terminally destructive for lymphocytes but not damaging to OCC.
A fine needle aspiration biopsy was taken from three ovary carcinoma cancer patients
with pleural and ascitic metastases. The pleural effusions were incubated for one hour and
then sludge moved on smears. Cytological smears were fixed with a mixture of alcoholacetone in a 1:1 ratio for 10 min and air-dried and endogenous peroxidase was inactivated
with 1% sodium azide (Merck) for 15 min. Then smears were washed twice by water and left
for five minutes in Tris-NaCl buffer (pH 7.6). After that, the field for immunohistochemical
analysis was surrounded with a hydrophobic pen (DakoCytomation) before the application
of pig serum (Novocastra). The anti-PD-1 monoclonal antibodies (NCL-HPp, Novocastra),
directed against PD-1 antigens, were applied after incubation with serum (30 min at
room temperature), and the preparation was incubated for an hour at 37°С. At the end
of labeling with the first antibodies, the preparations were washed twice with the buffer
for five minutes and pigs biotinylated antibodies (DakoCytomation) directed against antiPD-1 antibodies were applied. The second antibody preparations (cover slip impression)
were incubated for 15 min at room temperature. The next step of the immunohistochemical
procedure was preceded by washing preparations in two shifts buffer; coating was for 10
min at room temperature in an imaging system that consisted of a soluble complex—avid
in and biotinylated horseradish peroxidase (DakoCytomation). The 3,3’-Diaminobenzidine (DAB) in the format of Novocastra was used as a substrate for
the manifestation of immunohistochemical reaction. Cover slip
impression was additionally stained with haematoxylin. Analysis of
the preparations (cover slip impression) was carried out using Leica
DM 4000 B optical microscope with the immersion objective (×100).
We visualized a total of 793 unbound pleural PD-1+
T-cells and PD-1+
-
OCC aggregates.
Figure 1: Intact PD-1+
T-cells (1) and OCC (2)
Figure 2: Damaged PD-1+
T-cells (1) phagocytized by OCC (2)
Figure 3: Damaged PD-1+ T-cells (1) phagocytized by OCC (2) and
intact T-cells (3)