Abstract 2514: Cell-free chromatin particles released into the microenvironment from dying tumor cells activate cancer hallmarks in surviving cells: a study in advanced squamous cell carcinoma of oral cavity with therapeutic implications
Authors: Mittra Indraneel , Singhavi Hitesh , Pilankar Aishwarya , Gorantla Venkat Raghuram , Siddiqui Sophiya , Khare Naveen Kumar , Jadhav Vishalkumar , Pal Kavita , Chaturvedi Pankaj 
Published: July 2021

Abstract
Background: We have reported that combination of the nutraceuticals Resveratrol (R) and Copper(Cu) results in generation of free radicals which can degrade cell-free chromatin (cfCh) particles and have potential therapeutic effects (Mittra et al., Ann. Oncol. 2017 & Mittra et al., PLos One 2020). We have also reported that cfCh particles that are released from dying tumor cells can integrate into genomes of healthy bystander cells to activate two critical hallmarks of cancer namely, genome instability and inflammation, which could lead to their oncogenic transformation (Mittra et al., Cell Death Discovery 2017). Here we show that cfCh particles released into tumor microenvironment (TME) from dying tumor cells of advanced squamous carcinoma of oral cavity activate all ten hallmarks of cancer defined by Hanahan and Weinberg, and that oral administration of R-Cu for two weeks prevents this potentially life-threatening manifestation.
Patients and Methods: The study was approved by Institutional Ethics Committee, and a written consent was obtained from participants. In this exploratory study, R-Cu was administered in four escalating doses orally for two weeks, to 25 patients divided into 5 groups with 5 patients each, as follows: 1) Control: R 0, Cu 0; 2) Level I: R 5.6 mg, Cu 560 ng; 3) Level II: R 50 mg, Cu 5µg; 4) Level III: R 500 mg, Cu 50 µg; 5) Level IV: R 500 mg, Cu 5mg. A punch biopsy was obtained on the day 0 and day 14 post R-Cu treatment. FFPE histological sections were examined by 1) Fluorescence immuno-staining (FI) using anti-DNA and anti-histone H4 antibodies and confocal microscopy to detect presence of cfCh in TME; 2) Immuno-fluorescence (IF) analysis of intracellular antioxidant enzymes; and 3) IF analysis of 10 hallmarks of cancer represented by 23 biomarkers.
Results: 1) FI and Confocal microscopy detected extensive presence of dually fluorescently labelled cfCh particles in TME, which were dramatically reduced following R-Cu treatment. 2) IF analysis detected significantly elevated levels of three intra-cellular anti-oxidant enzymes in post R-Cu samples, suggesting that cells
had mounted a defense mechanism to counteract free radicals that had entered into them. No difference between day 0 and day 14 samples was seen in the untreated control group. 3) IF analysis of 23 cancer hallmark biomarkers showed significant down-regulation in the post R-Cu samples, which were most marked in dose levels I and II. No difference between day 0 and day 14 samples was seen in the control group.
Conclusion: We show in a human tumor model that cfCh particles released into TME from dying tumor cells activate cancer hallmarks in surviving cells, and that the cfCh inactivating agent R-Cu can significantly down-regulate cancer hallmark levels suggesting its therapeutic potential.
Citation Format: Indraneel Mittra, Hitesh Singhavi, Aishwarya Pilankar, Venkat Raghuram Gorantla, Sophiya Siddiqui, Naveen Kumar Khare, Vishalkumar Jadhav, Kavita Pal, Pankaj Chaturvedi. Cell-free chromatin particles released into the microenvironment from dying tumor cells activate cancer hallmarks in surviving cells: a study in advanced squamous cell carcinoma of oral cavity with therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2514.