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. 2024 Apr 5;12(4):809.
doi: 10.3390/biomedicines12040809.

PBMCs as Tool for Identification of Novel Immunotherapy Biomarkers in Lung Cancer

Caterina De Rosa  1 , Francesca Iommelli  2 , Viviana De Rosa  2 , Giuseppe Ercolano  3 , Federica Sodano  3 , Concetta Tuccillo  1 , Luisa Amato  1 , Virginia Tirino  4   5 , Annalisa Ariano  1 , Flora Cimmino  6 , Gaetano di Guida  1 , Gennaro Filosa  1 , Alessandra di Liello  1 , Davide Ciardiello  7 , Erika Martinelli  1 , Teresa Troiani  1 , Stefania Napolitano  1 , Giulia Martini  1 , Fortunato Ciardiello  1 , Federica Papaccio  8 , Floriana Morgillo  1 , Carminia Maria Della Corte  1
Affiliations

PBMCs as Tool for Identification of Novel Immunotherapy Biomarkers in Lung Cancer

Caterina De Rosa et al. Biomedicines. .

Abstract

Background: Lung cancer (LC), including both non-small (NSCLC) and small (SCLC) subtypes, is currently treated with a combination of chemo- and immunotherapy. However, predictive biomarkers to identify high-risk patients are needed. Here, we explore the role of peripheral blood mononuclear cells (PBMCs) as a tool for novel biomarkers searching.

Methods: We analyzed the expression of the cGAS-STING pathway, a key DNA sensor that activates during chemotherapy, in PBMCs from LC patients divided into best responders (BR), responders (R) and non-responders (NR). The PBMCs were whole exome sequenced (WES).

Results: PBMCs from BR and R patients of LC cohorts showed the highest levels of STING (p < 0.0001) and CXCL10 (p < 0.0001). From WES, each subject had at least 1 germline/somatic alteration in a DDR gene and the presence of more DDR gene mutations correlated with clinical responses, suggesting novel biomarker implications. Thus, we tested the effect of the pharmacological DDR inhibitor (DDRi) in PBMCs and in three-dimensional spheroid co-culture of PBMCs and LC cell lines; we found that DDRi strongly increased cGAS-STING expression and tumor infiltration ability of immune cells in NR and R patients. Furthermore, we performed FACS analysis of PBMCs derived from LC patients from the BR, R and NR cohorts and we found that cytotoxic T cell subpopulations displayed the highest STING expression.

Conclusions: cGAS-STING signaling activation in PBMCs may be a novel potential predictive biomarker for the response to immunotherapy and high levels are correlated with a better response to treatment along with an overall increased antitumor immune injury.

Keywords: PBMC; biomarker; cGAS-STING.

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Conflict of interest statement

Dr. Morgillo F.: receipt of honoraria or consultation fees for speaker, consultancy, or advisory roles: Roche, Servier, Incyte, ESMO and MSD. Dr. Papaccio F.: private research funding from Merck, travel support from Diatech Pharmacogenetics and recipient of an ESMO Research Fellowship sponsored by Amgen from 2018 to 2020. Dr Ciardiello D.: travel support from Sanofi, BMS and Merck KgA. Dr. Martinelli E.: receipt of honoraria or consultation fees for speaker, consultancy or advisory roles: Amgen, Bayer, Eisai, Merck Serono, Pierre Fabre, Roche, Servier, Incyte, ESMO, MSD; travel grant: AstraZeneca, Pierre Fabre. Dr. Troiani T.: receipt of honoraria or consultation fees for speaker, consultancy, or advisory roles: Merck, Amgen, Pierre Fabre, Servier, MSD, Bayer and Novartis. Dr. Napolitano S.: received travel grants from Amgen, Merck KGaA, Bristol Myers Squibb and Novartis outside the submitted work. Dr Martini G.: reported receiving honoraria from Servier and Incyte outside the submitted work. Dr. Ciardiello F.: receipt of honoraria or consultation fees for speaker, consultancy, or advisory roles: Amgen, Merck KGaA, MSD, Pierre Fabre, Pfizer, Roche Servier; institutional financial interests, financial support for clinical trials or contracted research; Amgen, Merck KGaA, MSD, Pierre Fabre, Pfizer, Roche, Servier. Dr. Della Corte C.M.: reported receiving personal fees from Roche, MSD and AstraZeneca, and travel grants from Amgen outside the submitted work. The remaining authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
A graphical summary of the PBMC isolation method step-by-step. The graphical scheme was produced by the authors using the free BioRender platform.
Figure 2
Figure 2
Real-time PCR analysis of in vitro STING, c-GAS, CXCL10 and CCL5 mRNA expression in lung cancer (LC) patient-derived PBMCs. Changes in mRNA levels were normalized to the expression of housekeeping genes (18S). Data are expressed as means ± SEM derived from n = 2 technical calculated by the comparative method 2−∆∆Ct. One-tailed unpaired Student’s t-test with CI = 95%, **** p < 0.0001.
Figure 3
Figure 3
Levels of CXCL10/IP-10 and CCL5 in LC patients’ serum were determined using ELISA assay. Each experiment was performed in duplicate. Data represent mean concentrations ± SD. One-tailed unpaired Student’s t-test with CI = 95%, **** p < 0.0001.
Figure 4
Figure 4
Gene name, variant types and number of variants of pathogenic DDR germline variants found in LC patients. Type and the therapy response of the corresponding LC groups are shown.
Figure 5
Figure 5
The mRNA expression levels of STING, cGAS, CXCL10 and CCL5 were studied by RT-qPCR in LC patient-derived PBMCs cultured in complete media supplemented with 10% HS for 24 h and then treated with DNA-PK-I 2 µM for 72 h; 18S was used as the reference gene. Results are expressed as means ± SEM derived from n = 2 technical calculated by the comparative method 2−∆∆Ct. One-tailed unpaired Student’s t-test with CI = 95%, **** p < 0.0001, ** p < 0.05.
Figure 6
Figure 6
(A–C) Representative immunofluorescence images showing the infiltration ability of immune cells in 3D tumor spheroids of H460 (A), H661 (B) and A549 (C) cells and evaluation of the red/blue intensity ratio as a quantitative indication of the co-localization rate of LC cells and PBMC. Statistical significance: **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. Three-dimensional tumor spheroids were blue stained, and the LC patient-derived PBMCs were red stained. The PBMCs were pre-treated with DNA-PK-I (2 µM) or cisplatin (0.5 µM) for 72 h and then added to 3D spheroids to start the co-culture. The co-localization of LC cells and PBMCs is reported under the merge column. Scale bar 100 µm.
Figure 7
Figure 7
(A) FACS analysis of LC patient-derived PBMC subset (upper panel) and % of STING-positive cells (lower panel). Statistical significance: ** p < 0.001, * p < 0.05. (B) Luminex cytokine assay of granzyme A, granzyme B, granulysin and perforin in LC patient-derived PBMC culture supernatant. **** p < 0.0001, *** p < 0.001, * p < 0.05.
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