Breast cancer is now globally the most frequent cancer and leading cause of women’s death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ERα-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ERα + breast cancer models. The ERα + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ERα signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK.

Globally, breast cancer is among the most diagnosed cancer types for women. Current and upcoming breast cancer therapies are being investigated in combination with compounds that stimulate an immune response, but whether the therapeutic agents themselves have unexpected immunomodulatory effects is often overlooked. Here, we have developed a method to grow 3D cultures of intact fragments of patient-derived tissue (Patient-Derived Explant Cultures; PDECs) to assess the preclinical potential of studying human tumor cells and immune cells simultaneously ex vivo Single cell sequencing, flow cytometry, gene expression profiling and cytokine profiling data show that the tumor immunocontexture is conserved in PDECs and that these resident immune cells respond to distinct immune stimulus We performed gene expression profiling, flow cytometry, and cytokine profiling of drug-treated human explants and found that metformin has antitumor potential through the activation of antigen presenting cells. We further validated in vitro that metformin-mediated APC activation is largely through mitochondrial respiration inhibition irrespective of the presence of tumor cells. Our PDEC platform highlights the preclinical potential of ex vivo explants by simultaneously offering information of tumor and immune cell toxicity and mechanism. Citation Format: Rita J. Turpin, Ruixian Liu, Pauliina Munne, Aino Peura, Jenna Rannikko, Gino Philips, Natasha Salmelin, Elina Hurskainen, Ilida Suleymanova, Minna Mutka, Tuomo Meretoja, Johanna Mattson, Satu Mustjoki, Päivi Saavalainen, Diether Lambrechts, Jeroen Pouwels, Maija Hollmén, Juha Klefström. TIL-containing patient-derived explant cultures reveal role of metformin on antigen presenting cell activation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4122.

Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. Here we establish a Patient-Derived Explant Culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+ T cell cytotoxic activity. We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax + metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+ T cells and thus facilitating anti-tumor immunity. Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.

Three-dimensional ex vivo cultures from patient-derived tumor tissue offer new opportunities for drug development and personalized treatment of cancer. However, one of the main challenges in establishing a tumor type faithful ex vivo tissue culture system is in optimization of biochemical and physical microenvironment to preserve desired phenotypic features. A collaboration with the Helsinki University Hospital enables us to receive live patient-derived breast cancer tissue samples from most breast cancer surgeries performed in the Southern Finland, typically samples arriving twice a week. Together with a group of materials scientists from Aalto Technical University we have explored seven different 3D culture matrices and defined their biomechanical properties and stiffness values with rheological measurements. Four investigated matrices preserved the luminal CK8+ cell phenotype of the luminal cancers they derived from, whereas rest of the matrices that included commonly used Matrigel and collagen scaffolds promoted a switch in the cell identity from luminal CK8+ to basal CK14+ phenotype. Furthermore, the genetic expression profiles examined with NGS corresponded to observed phenotypic switch. We noticed significant species-specific differences between mouse mammary and human breast samples in their morphology, phenotypic responses and ERα+ preservation to microenvironmental matrix stiffness. Furthermore, a combination of scaffold stiffness and composition of biochemical matrix constituency is required for long-term maintenance of ERα+ luminal tumour phenotype. Therefore, both physical properties and composition of the microenvironment need to be optimized for maintenance of breast tumor type specific phenotype and genetic profiles of ex vivo tumor explant cultures. Citation Format: Pauliina M. Munne, Lahja Martikainen, Iiris Räty, Kia Bertula, Nonappa Nonappa, Aino Peura, Minna Mutka, Marjut Leidenius, Tuomo Meretoja, Panu Kovanen, Johanna Mattson, Paivi Heikkila, Heikki Joensuu, Olli Ikkala, Juha T. Klefstrom. Novel ex vivo model for ERα positive breast cancer [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 2966.