乳腺癌（BC）是全球癌症死亡的主要原因之一。环磷酰胺（CTX）尽管有害不良反应和细胞死亡耐受性，仍然是癌症治疗的主要支柱。为了应对这种情况，化疗和免疫疗法的联合治疗已被提出。IMMUNEPOTENT CRP（ICRP）是一种免疫疗法，对多种癌细胞具有细胞毒性作用，而不影响外周血单核细胞（PBMC）和CD3 +细胞。本研究旨在评估CTX与ICRP（ICRP + CTX）在乳腺癌细胞中的细胞毒性，细胞毒性类型以及涉及细胞死亡的几个特征，以及它们对健康细胞的影响。为此，人类和小鼠乳腺癌细胞MCF-7，MDA-MB-231和4T1，或PBMC被用ICRP，CTX或ICRP + CTX处理24小时以评估细胞死亡。流式细胞术和显微镜技术用于确定细胞死亡的生化和形态学特征。实验表明，ICRP与CTX相结合诱导了强化的细胞死亡，表现为形态学改变，线粒体膜电位丧失，反应性氧（ROS）产生和Caspase活化。此外，确定ICRP + CTX引起的细胞死亡在所有评估的乳腺癌细胞中都是非Caspase依赖性的。另一方面，ICRP不影响PBMC中CTX的细胞毒性。基于以上所述，我们可以提出ICRP与CTX的组合疗法具有有效性，并推动其在蛋白质失调的肿瘤细胞中的应用，进一步推动ICRP与CTX联合治疗的发展。版权所有 © 2023 Rivera-Lazarín等。

Breast cancer (BC) is one of the leading causes of cancer death worldwide. Cyclophosphamide (CTX) remains a mainstay in cancer therapy despite harmful adverse effects and cell death-resistances. To face this, combinational therapy of chemotherapies and immunotherapies has been proposed. IMMUNEPOTENT CRP (ICRP) is an immunotherapy that has cytotoxic effects in several cancer cells without affecting peripheral blood mononuclear cells (PBMC) and CD3+ cells. The aim of this study was to evaluate cytotoxicity, the type of cytotoxic effect, and several features involved in cell death induced by the combination of CTX with ICRP (ICRP+CTX) in breast cancer cells as well as their effect on healthy cells. For this purpose, human and murine breast cancer cells, MCF-7, MDA-MB-231 and 4T1, or PBMC were treated for 24 hours with ICRP, CTX or ICRP+CTX in different combination ratios for the assessment of cell death. Flow cytometry and microscopy were used to determine biochemical and morphological characteristics of cell death. Assays showed that ICRP in combination with CTX induce potentiated cell death manifested with morphological changes, loss of mitochondrial membrane potential, reactive oxygen species (ROS) production, and caspase activation. In addition, it was determined that ICRP+CTX-cell death is caspase-independent in all the breast cancer cells assessed. On the other hand, ICRP did not affect CTX-cytotoxicity in PBMC. For all the above, we can propose that the combination of ICRP with CTX an effective combination therapy, promoting their use even in tumoral cells with defects on proteins implicated in the apoptotic pathway.Copyright © 2023 Rivera-Lazarín et al.