Caprine Herpes Virus-1 (CpHV-1) Reduces Cell Viability and Enhances Chemosensitivity in Breast Cancer Cells

Carmelina Antonella Iannuzzi¹Francesco Pagano²Marianna Tomeo^3,4Anna Sfera⁵Alessandra Calabrese¹Luigi Alfano¹Rosa Carmerlingo⁶Stefania Cocco¹Sara Damiano⁷Serena Montagnarp⁷ROBERTO CIARCIA⁷Antonio Giordano⁸Michelino De Laurentiis^1*Claudia Von Arx¹Iris Maria Forte^1,9*

• ¹Experimental Clinical Oncology of Breast Unit, Department of Breast and Thoracic Oncology, Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Naples, Italy, Naples, Italy
• ²Universita degli studi della Campania Luigi Vanvitelli Dipartimento di Salute Mentale e Fisica e Medicina Preventiva, Naples, Italy
• ³Scuola Superiore Meridionale, Naples, Italy
• ⁴Clinical and Translational Oncology Program, Scuola Superiore Meridionale (SSM, School of Advanced Studies), University of Naples Federico, Naples, Italy
• ⁵Universita degli Studi di Siena Dipartimento di Biotecnologie Mediche, Siena, Italy
• ⁶Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori, IRCCS, Fondazione G. Pascale, I-80131 Naples, Italy;, Naples, Italy
• ⁷Universita degli Studi di Napoli Federico II Dipartimento di Medicina Veterinaria e Produzioni Animali, Naples, Italy
• ⁸Temple University Sbarro Institute for Cancer Research and Molecular Medicine, Philadelphia, United States
• ⁹G. Pascale National Cancer Institute Foundation (IRCCS), Naples, Italy

Oncolytic viruses (OVs) are promising therapeutic agents in oncology that directly lyse tumor cells, modulate the immune response, and alter the tumor microenvironment. Non-human OVs offer advantages over their human counterparts, such as being non-pathogenic in humans and lacking pre-existing immunity. In previous studies, we demonstrated that a non-human caprine herpesvirus 1 (CpHV-1) effectively kills various human cancer cell lines. In this study, we evaluate CpHV-1's antitumor effects across different breast cancer (BC) cell lines and its potential synergy with FDA-approved BC therapies. We assessed the effects of CpHV-1 on BC cell viability and clonogenic potential, cell cycle regulation, and apoptosis in MCF-7, T47D, SKBR3 and MDA-MB-468 cell lines and on the non-tumorigenic mammary epithelial cells (MCF-10A). Additionally, CpHV-1 was tested in combination with Abemaciclib, Tucatinib, and Inavolisib, and synergism was evaluated using Chou-Talalay analysis. Our data show that CpHV-1 induced a dose-dependent cytotoxic effect, with an MOI of 5 reducing viability by ~50% 72 hours post-infection. Clonogenic assays confirmed long-term growth inhibition. We also demonstrated modulation of cell cycle progression and induction of apoptosis in tumour cells mediated by CpHV-1. Finally, combined treatments showed synergy across all BC subtypes, without significant toxicity in normal cells. These findings highlight CpHV-1 as a promising oncolytic agent, particularly in combination with targeted therapies, warranting further investigation for BC treatment.